Methods for reducing filter fouling in perfusion cell cultures with recirculating tangential flow filtration

Abstract

The present disclosure provides methods for producing recombinant proteins (e.g., antibodies) in mammalian cell culture under conditions that limit filter fouling and enable robust recirculating tangential flow (RTF) filtration operation in perfusion processes in which the working volume is increased to a final working volume during the cell culture.

Description

METHODS FOR REDUCING FILTER FOULING IN PERFUSION CELL CULTURES WITH RECIRCULATING TANGENTIAL FLOW FILTRATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 672,182, filed July 16, 2024.FIELD OF THE INVENTION

[0002] The present disclosure relates to perfusion cell culture processes for producing recombinant proteins, more particularly to methods for improving recirculating tangential flow (RTF) filtration performance in perfusion processes employing a feed-up step by adding antifoam to reduce filter fouling.BACKGROUND OF THE INVENTION

[0003] As the demand for therapeutic recombinant proteins continues to grow, much effort is being placed on process optimization, particularly strategies for growing, feeding, and maintaining production cell cultures which have a positive impact on cell viability and protein production. Developing manufacturing processes for the production of recombinant proteins is a complex endeavor in which many variables must be balanced. This is particularly true for upstream processes, where every element of the cell culture process can have a large impact on the later stages of production.

[0004] Mammalian cells are often used to produce therapeutic proteins. In some processing methods, mammalian cells are cultured in a perfusion bioreactor system where a volume of cell culture containing the cell culture waste is removed from the bioreactor, and new culture medium is added to replace the volume removed. In such perfusion culturing systems, the removed cell culture is often filtered to retain the mammalian cells in the bioreactor for further recombinant protein production, while the culture medium (sometimes referred to as “spent medium”) containing a recombinant protein can be recovered at the end of the process. On balance, it has been recognized that perfusion culture offers relatively good economics for cell cultures. Perfusion culture operation can achieve high cell densities and, more importantly, the cells can be maintained in a highly productive state for weeks. Accordingly, perfusion-based cell culture can achieve much higher yields and reduce the size of the bioreactor necessary relative to fed batch and batch operations.

[0005] Filtration is critical in perfusion processes because of the continuous flow of cell culture material and byproducts through the fdter. Filtration approaches for cell culture commonly use tangential flow fdtration (also known as TFF). In TFF, fluid to be filtered is circulated with a pump, typically, from a reservoir through a filter and back to the reservoir. See, e.g., U.S. Patent No. 9,663,753. Recirculating tangential flow (RTF) and alternating tangential flow (ATF) filtration systems represent two distinct approaches to tangential flow filtration in perfusion cell culture applications. When the cell culture is recirculated through the filtration membrane, it is known as recirculating tangential flow (RTF) filtration. See, e.g., U.S. Patent Application Publication Nos. US20160222337 and US20140093952. Specifically, cell culture fluid is continuously circulated through the filter in a unidirectional flow pattern in RTF systems, creating a consistent crossflow across the membrane surface. The fluid enters the filter, passes tangentially across the membrane, and exits as retentate that returns to the bioreactor, while permeate passes through the membrane pores. This continuous circulation is typically driven by peristaltic or centrifugal pumps that maintain steady flow rates. Alternating tangential flow (ATF) filtration, which employs a diaphragm pump mechanism that creates an alternating, bidirectional flow pattern across the membrane surface, offers an alternative mode of filtration. See, e.g., U.S. Patent No. 10,081,788. ATF is similar in some respects to RTF as it generates a flow pattern parallel to the filtration membrane surface. However, ATF differs from RTF in that the direction of flow is repeatedly alternating or reversing across the filter surface through the use of a diaphragm pump and rapid alternating cycling (on the order of ~10 seconds). While ATF and RTF systems are both employed in bioprocessing, the filtration efficiency of these systems is not optimal, and they are sensitive to clogging and fouling of the membranes.

[0006] Although ATF filtration is commonly used in biomanufacturing to perform perfusion during Ultra-Filtration (UF) and harvest during Microfiltration (MF), ATF system performance may suffer at the larger scales that are increasingly being employed in intensified processes, particularly with higher cell densities and packed cell volumes, where the alternating flow pattern may lead to coconcentration of cells and debris within the system.

[0007] RTF filtration is being evaluated as a technology for performing perfusion cell culture in support of efforts to reduce cost of sales (COS) for the production of high-volume biologies. RTF systems may offer advantages in terms of facility integration and scalability relative to ATF systems as well as other potential benefits for RTF. For example, RTF filtration can offer better control overflow rates and shear conditions, enabling scale up, as well as reduced fouling compared to ATF filtration due to continuous sweeping of the membrane surface. However, previous work to evaluate the use of RTF filtration on a production bioreactor (N-0) for such high-volume biologies produced, for example, using perfusion processes in which the working volume is increased to a final working volume during the cell culture, has proven problematic due to recurring cellular debris accumulation at the inlet of the filter.

[0008] Accordingly, there is a need for improved methods for RTF filtration for perfusion processes, including methods that reduce clogging at the filter inlet of the RTF filtration system in perfusion processes employing a feed-up step.SUMMARY OF THE INVENTION

[0009] The present disclosure provides in an embodiment 1, a method for producing a recombinant protein comprising: a) culturing mammalian cells expressing a recombinant protein in a bioreactor under perfusion and recirculating tangential flow (RTF) filtration conditions sufficient to produce the recombinant protein and at a working volume from 50% to 95% of the final working volume; b) maintaining the foam coverage at the fluid surface of the bioreactor at a target of 10% or less through the presence of antifoam; c) increasing the working volume to the final working volume wherein the cell culture within the bioreactor contains antifoam in an amount of at least 14 mg antifoam / kg of cell culture (mg / kg) at the initiation of increasing the working volume, wherein the amount of antifoam is calculated based on the final working volume; and d) culturing the mammalian cells for the duration of a production run.

[0010] In a sub-embodiment of embodiment 1, the amount of antifoam at step c) is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is at least 15 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is at least 16 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is at least 17 mg / kg. In some subembodiments, the amount of antifoam at step c) is at least 18 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is at least 19 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is at least 20 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is at least 21 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is at least 22 mg / kg. In somesub-embodiments, the amount of antifoam at step c) is at least 23 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is at least 24 mg / kg.

[0011] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments thereof, the amount of antifoam at step c) is no more than 37, 38, 39, 40, 45, 50, or 60 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is no more than 37 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is no more than 38 mg / kg. In some subembodiments, the amount of antifoam at step c) is no more than 39 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is no more than 40 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is no more than 45 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is no more than 50 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is no more than 60 mg / kg.

[0012] In some sub-embodiments, the amount of antifoam at step c) is in the range of 15 mg / kg to 37 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 15 mg / kg to 38 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 15 mg / kg to 39 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 15 mg / kg to 40 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 15 mg / kg to 45 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 15 mg / kg to 50 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 15 mg / kg to 60 mg / kg.

[0013] In some sub-embodiments, the amount of antifoam at step c) is in the range of 16 mg / kg to 37 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 16 mg / kg to 38 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 16 mg / kg to 39 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 16 mg / kg to 40 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 16 mg / kg to 45 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 16 mg / kg to 50 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 16 mg / kg to 60 mg / kg.

[0014] In some sub-embodiments, the amount of antifoam at step c) is in the range of 17 mg / kg to 37 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 17 mg / kg to 38 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 17 mg / kgo 39 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 17 mg / kgo 40 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 17 mg / kgo 45 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 17 mg / kgo 50 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 17 mg / kgo 60 mg / kg.

[0015] In some sub-embodiments, the amount of antifoam at step c) is in the range of 18 mg / kgo 37 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 18 mg / kgo 38 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 18 mg / kgo 39 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 18 mg / kgo 40 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 18 mg / kgo 45 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 18 mg / kgo 50 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 18 mg / kgo 60 mg / kg.

[0016] In some sub-embodiments, the amount of antifoam at step c) is in the range of 19 mg / kgo 37 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 19 mg / kgo 38 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 19 mg / kgo 39 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 19 mg / kgo 40 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 19 mg / kgo 45 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 19 mg / kgo 50 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 19 mg / kgo 60 mg / kg.

[0017] In some sub-embodiments, the amount of antifoam at step c) is in the range of 20 mg / kgo 37 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 20 mg / kgo 38 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 20 mg / kgo 39 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 20 mg / kgo 40 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 20 mg / kgo 45 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 20 mg / kgo 50 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 20 mg / kgo 60 mg / kg.

[0018] In some sub-embodiments, the amount of antifoam at step c) is in the range of 21 mg / kgo 37 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 21 mg / kgo 38 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 21 mg / kgo 39 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 21 mg / kgo 40 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 21 mg / kgo 45 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 21 mg / kgo 50 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 21 mg / kgo 60 mg / kg.

[0019] In some sub-embodiments, the amount of antifoam at step c) is in the range of 22 mg / kgo 37 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 22 mg / kgo 38 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 22 mg / kgo 39 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 22 mg / kgo 40 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 22 mg / kgo 45 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 22 mg / kgo 50 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 22 mg / kgo 60 mg / kg.

[0020] In some sub-embodiments, the amount of antifoam at step c) is in the range of 23 mg / kgo 37 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 23 mg / kgo 38 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 23 mg / kgo 39 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 23 mg / kgo 40 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 23 mg / kgo 45 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 23 mg / kgo 50 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 23 mg / kgo 60 mg / kg.

[0021] In some sub-embodiments, the amount of antifoam at step c) is in the range of 24 mg / kgo 37 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 24 mg / kgo 38 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 24 mg / kgo 39 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 24 mg / kgo 40 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 24 mg / kgo 45 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 24 mg / kgto 50 mg / kg. In some sub-embodiments, the amount of antifoam at step c) is in the range of 24 mg / kg to 60 mg / kg.

[0022] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments thereof, the culturing in step a) is with a bioreactor inoculated with 1 x 106to 20 x 106cells / ml, 2 x 106to 18 x 106cells / ml, 4 x 106to 16 x 106cells / ml, 6 x 106to 14 x 106cells / ml, or 8 x 106to 12 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 1 x 106to 20 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 2 x 106to 18 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 4 x 106to 16 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 6 x 106to 14 x 106cells / ml. In some subembodiments, the culturing in step a) is with a bioreactor inoculated with 8 x 106to 12 x 106cells / ml.

[0023] In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 1 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 2 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 4 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 6 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 8 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 12 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 14 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 16 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 18 x 106cells / ml. In some sub-embodiments, the culturing in step a) is with a bioreactor inoculated with 20 x 106cells / ml.

[0024] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments thereof, the increasing the working volume step is initiated between 24 to 120 hours or 48 to 96 hours after initiation of the culturing in step a). In some sub-embodiments, the increasing the working volume step is initiated between 24 to 120 hours after initiation of the culturing in step a). In some sub-embodiments, the increasing the working volume step is initiated between 48 to 96 hours after initiation of the culturing in step a). In some sub-embodiments, the increasing the working volume step is initiated between 48 to 96 hours after initiation of the culturing in step a). In some subembodiments, the increasing the working volume step is initiated between 60 to 84 hours afterinitiation of the culturing in step a). In some sub-embodiments, the increasing the working volume step is initiated between 66 to 78 hours after initiation of the culturing in step a). In some subembodiments, the increasing the working volume step is initiated between 68 to 76 hours after initiation of the culturing in step a). In some sub-embodiments, the increasing the working volume step is initiated between 70 to 74 hours after initiation of the culturing in step a).

[0025] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments thereof, the increasing the working volume step is maintained for 24 to 48 hours. In some sub-embodiments, the increasing the working volume step is maintained for 24 to 48 hours. In some sub-embodiments, the increasing the working volume step is maintained for 28 to 36 hours. In some sub-embodiments, the increasing the working volume step is maintained for 26 to 34 hours. In some sub-embodiments, the increasing the working volume step is maintained for 25 to 32 hours. In some sub-embodiments, the increasing the working volume step is maintained for 27 to 31 hours.

[0026] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments thereof, the culturing in step a) is at a working volume from 60% to 70% of the final working volume. In some sub-embodiments, the culturing in step a) is at a working volume from 65% to 70% of the final working volume.

[0027] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments and aspects thereof, the recirculating tangential flow filtration is through a hollow fiber filter, a flat sheet membrane, or a spiral -wound membrane. In one aspect of this sub-embodiment, the recirculating tangential flow filtration is through a hollow fiber filter. In another aspect of this subembodiment, the recirculating tangential flow filtration is through a flat sheet membrane. In another aspect of this sub-embodiment, the recirculating tangential flow filtration is through a spiral-wound membrane. In certain aspects of this sub-embodiment, the hollow fiber filter, flat sheet membrane, or spiral-wound membrane has a pore size or molecular weight cut off that retains the recombinant protein and cells in the bioreactor. In certain aspects, the hollow fiber filter, flat sheet membrane, or spiral-wound membrane has a pore size or molecular weight cut off that retains the recombinant protein and cells in the bioreactor is an ultrafilter. In certain sub-aspects, the molecular weight cutoff of the ultrafilter is 50 kDa and / or the pore size of the ultrafilter is 0.01 micrometers to 0. 1 micrometers.

[0028] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments and aspects thereof, antifoam is added after the increasing the working volume step in an amount sufficient to maintain less than 50% foam coverage at the fluid surface of the bioreactor. In one aspect of this sub-embodiment, antifoam is added after the increasing the working volume step in an amount sufficient to maintain less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% foam coverage at the fluid surface of the bioreactor. In some sub-embodiments, antifoam is added after the increasing the working volume step in an amount sufficient to maintain less than 30% foam coverage at the surface of the bioreactor. In some sub-embodiments, antifoam is added after the increasing the working volume step in an amount sufficient to maintain less than 25% foam coverage at the surface of the bioreactor. In some sub-embodiments, antifoam is added after the increasing the working volume step in an amount sufficient to maintain less than 20% foam coverage at the surface of the bioreactor. In some sub-embodiments, antifoam is added after the increasing the working volume step in an amount sufficient to maintain less than 15% foam coverage at the surface of the bioreactor. In some sub-embodiments, antifoam is added after the increasing the working volume step in an amount sufficient to maintain less than 10% foam coverage at the surface of the bioreactor. In one aspect of this sub-embodiment, the amount of antifoam at the end of the culturing step d) is between 70 mg / kg and 300 mg / kg or between 80 mg / kg and 240 mg / kg. In some subembodiments, the amount of antifoam at the end of the culturing step d) is between 70 mg / kg and 300 mg / kg. In some sub-embodiments, the amount of antifoam at the end of the culturing step d) is between 80 mg / kg and 240 mg / kg. In some sub-embodiments, the amount of antifoam at the end of the culturing step d) is between 100 mg / kg and 150 mg / kg.

[0029] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments and aspects thereof, the antifoam is a silicone-based antifoam.

[0030] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments and aspects thereof, the antifoam is simethicone.

[0031] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments thereof, the antifoam is added in a continuous or semi-continuous manner or as a bolus. In some sub-embodiments, the antifoam is added in a continuous manner. In some sub-embodiments, the antifoam is added in a semi-continuous manner. In some sub-embodiments, the antifoam is added as a bolus. In an aspect of this sub-embodiment, the antifoam is added to the bioreactor separatelyfrom the perfusion culture media. In some sub-embodiments, the antifoam is simethicone and is added via a 1% simethicone solution.

[0032] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments and aspects thereof, the bioreactor volume ranges from 200L to 20000L. In some subembodiments, the bioreactor volume ranges from 200L to 5000L. In some sub-embodiments, the bioreactor volume ranges from 200L to 2000L. In some sub-embodiments, the bioreactor volume is 500L, WOOL, 2000L, or 5000L. In some sub-embodiments, the bioreactor volume is 200L. In some sub-embodiments, the bioreactor volume is 400L. In some sub-embodiments, the bioreactor volume is 500L. In some sub-embodiments, the bioreactor volume is 2000L. In some sub-embodiments, the bioreactor volume is 5000L. In some sub-embodiments, the bioreactor volume is 10000L. In some sub-embodiments, the bioreactor volume is 15000L. In some sub-embodiments, the bioreactor volume is 20000L.

[0033] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments and aspects thereof, the recombinant protein is an immunoglobulin, antibody, or antibody fragment. In some sub-embodiments, the recombinant protein is an immunoglobulin. In some sub-embodiments, the recombinant protein is an antibody. In some sub-embodiments, the recombinant protein is an antibody fragment.

[0034] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments and aspects thereof, the duration of the production run is 10 to 40 days, 10 to 20 days, or 10 to 15 days. In some sub-embodiments, the duration of the production run is 10 to 40 days. In some sub-embodiments, the duration of the production run is 10 to 20 days. In some subembodiments, the duration of the production run is 10 to 15 days.

[0035] In a sub-embodiment of embodiment 1, or a sub-embodiment of embodiment 1 and subembodiments and aspects thereof, the method further comprises e) harvesting the recombinant protein. In certain aspects of this sub-embodiment, harvesting includes filtering the permeate through a microfdtration (MF) fdter. In certain sub-aspects of this sub-embodiment, the MF filter has a MWCO of 750 kDa and / or the MF filter has a pore size of 0.2 pm.

[0036] The present disclosure also provides in an embodiment 2, a method for producing a recombinant protein comprising: a) culturing mammalian cells expressing a recombinant protein in abioreactor under perfusion and recirculating tangential flow (RTF) fdtration conditions sufficient to produce the recombinant protein and at a working volume from 65% to 75% of the final working volume; b) maintaining the foam coverage at the fluid surface of the bioreactor at a target of 10% or less through the presence of antifoam; c) increasing the working volume to the final working volume, wherein the cell culture within the bioreactor contains antifoam in an amount in the range of 20 to 40 mg antifoam / kg of cell culture (mg / kg) at the initiation of increasing the working volume, wherein the amount of antifoam is calculated based on the final working volume; and d) culturing the mammalian cells for the duration of a production run, wherein the amount of antifoam at the end of the culturing step d) is between 80 mg / kg and 240 mg / kg and the antifoam is simethicone.

[0037] The present disclosure further provides in an embodiment 3, a method for producing a recombinant protein comprising: a) culturing mammalian cells expressing a recombinant protein in a bioreactor under perfusion and recirculating tangential flow (RTF) filtration conditions sufficient to produce the recombinant protein and at a working volume from 65% to 75% of the final working volume; b) maintaining the foam coverage at the fluid surface of the bioreactor at a target of 10% or less through the presence of antifoam; c) increasing the working volume to the final working volume wherein the cell culture within the bioreactor contains antifoam in an amount in the range of 20 to 40 mg antifoam / kg of cell culture (mg / kg) at the initiation of increasing the working volume, wherein the amount of antifoam is calculated based on the final working volume and wherein the increasing is initiated 48 hours to 96 hours after initiation of the culturing in step a); and d) culturing the mammalian cells for the duration of a production run, wherein the amount of antifoam at the end of the culturing step d) is between 80 mg / kg and 240 mg / kg and the antifoam is simethicone.

[0038] The present disclosure further provides in an embodiment 4, a method for producing an antibody comprising: a) culturing Chinese hamster ovary (CHO) cells expressing an antibody in a bioreactor under perfusion and recirculating tangential flow (RTF) filtration conditions sufficient to produce the antibody and at a working volume from 65% to 75% of the final working volume; b) maintaining the foam coverage at the fluid surface of the bioreactor at a target of 10% or less through the presence of antifoam; c) increasing the working volume to the final working volume, wherein the cell culture within the bioreactor contains antifoam in an amount in the range of 20 to 40 mg antifoam / kg of cell culture (mg / kg) at the initiation of increasing the working volume, wherein the amount of antifoam is calculated based on the final working volume and wherein the increasing isinitiated 48 hours to 96 hours after initiation of the culturing in step a); and d) culturing the CHO cells for the duration of a production run, wherein the amount of antifoam at the end of the culturing step d) is between 80 mg / kg and 240 mg / kg and the antifoam is simethicone.

[0039] The present disclosure also provides in an embodiment 5, a method for producing a recombinant protein (e.g., an antibody) comprising: initiating a culture in a bioreactor at a working volume that is at least 50% of a final working volume; inoculating the culture with mammalian cells (e.g., CHO cells) engineered to express the recombinant protein; perfusing the culture at one or more perfusion rates of less than or equal to 0.5 culture volumes / day (V / d) until the culture reaches one or more desired target criteria, wherein foam coverage at the fluid surface of the bioreactor is maintained at 10% or less during the perfusing; increasing the working volume to the final working volume, wherein the bioreactor contains antifoam in an amount of at least 14 mg antifoam / kg of cell culture (mg / kg) at the initiation of increasing the working volume, wherein the amount of antifoam is calculated based on the final working volume; and once the final working volume is achieved, perfusing the culture at one or more perfusion rates of less than or equal to 0.5 V / d until the culture is terminated or harvested, wherein all rates expressed in V / d are calculated based on the final working volume. In some sub-embodiments, the amount of antifoam when the culture is terminated or harvested is between 80 mg / kg and 240 mg / kg and the antifoam is simethicone.

[0040] In some sub-embodiments of embodiment 5, the culture is initiated at a working volume that is 50% to 75% of the final working volume. In some sub-embodiments, the culture is initiated at a working volume that is 60% to 70% of the final working volume. In some sub-embodiments, the culture is inoculated at a cell density of 5 x 106cells / mL to 50 x 106cells / mL. In some subembodiments, the culture is inoculated at a cell density of 6 x 106cells / mL to 20 x 106cells / mL. In some sub-embodiments, the bioreactor is operated in batch mode for up to 24 hours following inoculation. In some sub-embodiments, the culture is in a growth phase prior to the increase in working volume to the final working volume. In some sub-embodiments, the culture is in a production phase following the increase in working volume to the final working volume. In some subembodiments, the culture is in a growth phase prior to the increase in working volume to the final working volume, and the culture is in a production phase following the increase in working volume to the final working volume. In some sub-embodiments, the one or more desired target criteria is time post-inoculation, wherein the time post-inoculation is 24 hours to 72 hours. In some sub-embodiments, the culture is perfused at one or more perfusion rates of 0.05 V / d to 0.5 V / d until the culture reaches one or more desired target criteria, wherein the feed rate(s) and the contemporaneous permeate rate(s) are the same. In some sub-embodiments, once the final working volume is achieved, the culture is perfused at one or more perfusion rates of 0.05 V / d to 0.5 V / d (e.g., 0. 10 V / d to 0.25 V / d) until the culture is terminated or harvested, wherein the feed rate(s) and the contemporaneous permeate rate(s) are the same. In some sub-embodiments, the culture is perfused at one or more perfusion rates of 0.05 V / d to 0.5 V / d (e.g., 0.10 V / d to 0.25 V / d) until the culture reaches one or more desired target criteria, wherein the feed rate(s) and the contemporaneous permeate rate(s) are the same, and, once the final working volume is achieved, the culture is perfused at one or more perfusion rates of 0.05 V / d to 0.5 V / d (e.g., 0.10 V / d to 0.25 V / d) until the culture is terminated or harvested, wherein the feed rate(s) and the contemporaneous permeate rate(s) are the same. In some subembodiments, the working volume is increased to the final working volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates, e.g., at least one of the one or more feed rates is less than or equal to 0.50 V / d (e.g., less than or equal to 0.40 V / d) and at least one of the one or more permeate rates is less than or equal to 0.20 V / d (e.g., less than or equal to 0.15 V / d), wherein each of the one or more feed rates is greater than the contemporaneous permeate rate.

[0041] The present disclosure also provides in an embodiment 6, a method for producing a recombinant protein (e.g., an antibody) comprising: initiating a culture in a bioreactor at a working volume that is at least 50% of a final working volume; inoculating the culture with mammalian cells (e.g., CHO cells) engineered to express the recombinant protein; perfusing the culture for 24 hours to 72 hours at one or more perfusion rates of 0.05 V / d to 0.5 V / d (e.g., 0.10 V / d to 0.25 V / d), wherein foam coverage at the fluid surface of the bioreactor is maintained at 10% or less during the perfusing; increasing the working volume to the final working volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates, wherein at least one of the one or more feed rates is less than or equal to 0.50 V / d (e.g., less than or equal to 0.4 V / d) and at least one of the one or more permeate rates is less than or equal to 0.20 V / d (e.g., less than or equal to 0. 15 V / d), wherein each of the one or more feed rates is greater than the contemporaneous permeate rate and further wherein the bioreactor contains simethicone in an amount of at least 14 mg simethicone / kg of cell culture (mg / kg) at the initiation of increasing the working volume, wherein theamount of simethicone is calculated based on the final working volume; and once the final working volume is achieved, perfusing the culture at one or more perfusion rates of less than or equal to 0.5 V / d until the culture is terminated or harvested, wherein the amount of simethicone when the culture is terminated or harvested is between 80 mg / kg and 240 mg / kg and further wherein all rates expressed in V / d are calculated based on the final working volume.BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIGs. 1A and IB provide schematic representations of (A) Recirculating Tangential Flow (RTF) and (B) Alternating Tangential Flow (ATF) filtration systems. The RTF system in FIG. 1A comprises a bioreactor containing cell culture fluid, a hollow fiber filter, and a pump that creates a unidirectional flow pattern, while the ATF system in FIG. IB comprises a bioreactor containing cell culture fluid, a hollow fiber filter, and a diaphragm pump that creates an alternating, bidirectional flow pattern across the membrane surface.

[0043] FIGs. 2A and 2B provide feed flow pressure over time for successful runs post-antifoam schedule implementation compared to runs pre-antifoam schedule implementation. FIG. 2A depicts feed flow pressure minus retentate pressure over time, while FIG. 2B shows the feed flow pressure over time. Pressure spikes were observed in pre-antifoam schedule implementation runs, which is indicative of filter fouling. In contrast, post-antifoam schedule implementation runs show stable pressure profiles without the dramatic spikes, demonstrating that the antifoam strategy effectively prevents filter fouling during the RTF process.

[0044] FIGs. 3A and 3B show (A) the total antifoam delivered per N-0 w.v. prior to feed-up initiation, as well as (B) the amount added over the N-0 process, for a series of successful (shown in dark grey) and unsuccessful RTF runs (shown in light grey) which experienced crossflow failures due to inlet debris accumulation. Successful RTF runs had higher initial antifoam concentrations (FIG.3A) and maintained higher antifoam levels throughout the cell culture process (FIG. 3B) compared to unsuccessful runs.

[0045] FIGs. 4A and 4B show (A) feed flow pressure and (B) permeate pressure over time for a series of RTF runs across multiple bioreactor scales (200L, 500L, and 2000L) in both stainless steel and single-use bioreactor formats. The depicted runs employed an RTF antifoam schedule describedherein to reduce foaming. The stable pressure profiles demonstrate that the antifoam strategy described herein enables robust RTF operation across a range of bioreactor scales and formats.

[0046] FIGs. 5A-5C show the following parameters for example ATF and RTF cultures over time: (A) viable cell density (VCD); (B) viability; and (C) PCV adjusted titer. VCD measurements showed comparable cell growth profiles for both ATF and RTF systems (FIG. 5A), with somewhat lower cell culture viability for RTF versus ATF (FIG. 5B), potentially due to different rates of cell shear associated with the RTF feed pump, but comparable overall productivity (FIG. 5C).DETAILED DESCRIPTION OF THE INVENTION

[0047] The present disclosure is based in part on the discovery that the addition of high amounts of an antifoam, such as simethicone, prior to feed-up, i.e., increasing the working volume to a final working volume, in a recirculating tangential flow (RTF) filtration system helps prevent clogging of the filter during perfusion cell culture processes that utilize such a feed-up step. Controlling foam buildup on the reactor is instrumental to RTF success as it has been found to prevent the accumulation of debris on the sidewalls of the bioreactor vessel. In such RTF systems, when foam is allowed to accumulate, cell debris remains at the fluid surface of the cell culture for an extended period, where it desiccates and forms a “crust layer.” When the feed-up begins, this crust layer is introduced into the bioreactor’s working volume and eventually accumulates at the inlet of the RTF filter, leading to catastrophic filter fouling. ATF is not as impacted by this “crust layer” due to the bidirectional flow of material through the HFM (Hollow Filter Membrane). FIGs. 1A and IB provide illustrations that compare the basic flow paths of ATF and RTF systems. Specifically, RTF filtration systems utilize a unidirectional recirculating flow pattern, while ATF filtration systems employ an alternating bidirectional flow pattern across the membrane surface.

[0048] Maintaining less than 10% foam coverage on the cell culture surface or fluid surface of the bioreactor prior to feed-up, or increasing the working volume to the final working volume, ensures that the RTF filter is free from clogging. It is important that some antifoam is present in the cell culture at the initiation of the cell culture or shortly thereafter. Generally, it is desirable to add or have present in the cell culture sufficient antifoam to maintain the foam coverage at the fluid surface of the bioreactor at a target of 10% or less. By “maintaining the foam coverage at the fluid surface of the bioreactor at a target of 10% or less through the presence of antifoam,” it is meant that whenever thefoam coverage at the fluid surface of the bioreactor approaches or exceeds 10%, antifoam is added to bring the foam coverage back down to below 10%. Thus, the foam coverage can increase to over 10% (e.g., up to 15%, up to 20%, or up to 25%) for a short time before it is brought back down to below 10%. To prevent the foam coverage from exceeding 10% or to increase the time it takes for the foam surface coverage to exceed 10%, antifoam can be present at the start of the cell culture.

[0049] In some embodiments of methods described herein, the foam coverage at the fluid surface of the bioreactor is maintained at a target of 9% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor is maintained at a target of 8% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor is maintained at a target of 7% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor is maintained at a target of 6% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor is maintained at a target of 5% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor is maintained at a target of 4% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor is maintained at a target of 3% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor is maintained at a target of 2% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor is maintained at a target of 1% or less.

[0050] The critical parameter for the methods described herein is that the antifoam present at the start of feed-up, or at increasing the working volume to the final working volume, be greater than or equal to about 14 mg of antifoam / kg of cell culture (mg / kg), wherein the amount of antifoam is calculated based on the final working volume. The amount of antifoam is calculated based on the mass of the cell culture at the final working volume, as measured by, for example, load cells. The mass (kg) of cell culture includes the cell bulk and cell culture media and is the total mass within the bioreactor. The mass can be measured by load cells or any other means known to those skilled in the art. In some embodiments, the mass of the cell culture is measured by load cells.

[0051] During feed-up, or increasing the working volume to the final working volume, the cell culture volume is allowed to increase through the addition of fresh cell culture media at a rate greater than the rate at which the spent cell culture media is removed. In certain embodiments, the level of antifoam at the start (i.e., initiation) of feed-up, or prior to increasing the working volume to the final working volume, is greater than or equal to 16 mg / kg, greater than or equal to 18 mg / kg, greater thanor equal to 20 mg / kg, greater than or equal to 22 mg / kg, greater than or equal to 24 mg / kg, greater than or equal to 26 mg / kg, greater than equal to 28 mg / kg, greater than or equal to 30 mg / kg, greater than or equal to 32 mg / kg, greater than equal to 34 mg / kg, greater than equal to 36 mg / kg, greater than equal to 38 mg / kg, or greater than equal to 40 mg / kg of cell culture. In certain sub-embodiments, the level of antifoam is less than or equal to 60 mg / kg of cell culture.

[0052] In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 16 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 18 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 20 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 22 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 24 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 26 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 28 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 30 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 32 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 34 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 36 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 38 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is greater than or equal to 40 mg / kg.

[0053] In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 16 mg / kg to 60 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 18 mg / kg to 60 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 20 mg / kg to 60 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 22 mg / kg to 60 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 24 mg / kg to 60 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 26 mg / kg to 60 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 28 mg / kg to 60 mg / kg. In someembodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 30 mg / kg to 60 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 32 mg / kg to 60 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 34 mg / kg to 60 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 36 mg / kg to 60 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 38 mg / kg to 60 mg / kg. In some embodiments, the level of antifoam at the start (i.e., initiation) of feed-up is in the range of 40 mg / kg to 60 mg / kg.

[0054] In methods disclosed herein, culturing mammalian cells expressing the recombinant protein is at a working volume from 50% to 95% of the final working volume. In certain subembodiments, culturing mammalian cells expressing the recombinant protein is at a working volume from 60% to 80% of the final working volume. In certain methods disclosed herein, a cell culture can be established or provided at a working volume from 50% to 95% of the final working volume in a bioreactor with mammalian cells expressing a recombinant protein. In certain embodiments, a cell culture can be established or provided at a working volume from 60% to 80% of the final working volume. In certain embodiments, the working volume is approximately 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, or 80% of the final working volume. For example, in some embodiments, the working volume is approximately 60% of the final working volume. In some embodiments, the working volume is approximately 62% of the final working volume. In some embodiments, the working volume is approximately 64% of the final working volume. In some embodiments, the working volume is approximately 66% of the final working volume. In some embodiments, the working volume is approximately 68% of the final working volume. In some embodiments, the working volume is approximately 70% of the final working volume. In some embodiments, the working volume is approximately 72% of the final working volume. In some embodiments, the working volume is approximately 74% of the final working volume. In some embodiments, the working volume is approximately 76% of the final working volume. In some embodiments, the working volume is approximately 78% of the final working volume. In some embodiments, the working volume is approximately 80% of the final working volume. Starting at a higher percentage of the final working volume can reduce the time of feed-up, but it is desirable tohave the cell culture at a working volume between 60% to 80% of the final working volume for optimal protein production.

[0055] A bioreactor is inoculated with mammalian cells expressing a recombinant protein to establish or provide a cell culture which is used for culturing. In certain sub-embodiments, the inoculated cell density is about 6 x 106cells / mL to about 50 x 106cells / mL, about 6 x 106cells / mL to about 25 x 106cells / mL, about 6 x 106cells / mL to about 20 x 106cells / mL, about 6 x 106cells / mL to about 15 x 106cells / mL, about 6 x 106cells / mL to about 14 x 106cells / mL, about 6 x 106cells / mL to about 13 x 106cells / mL, about 6 x 106cells / mL to about 12 x 106cells / mL, about 6 x 106cells / mL to about 11 x 106cells / mL, or about 6 x 106cells / mL to about 10 x 106cells / mL.

[0056] In the methods of the disclosure, a perfusion process is typically initiated within a day after inoculation (after a period of batch culture). In a perfusion process, the volume of the cell culture medium removed is approximately the same as the volume of the cell culture medium being added. As is known in the art, the rate at which the volume of the cell culture medium being removed (volume / unit of time) and the rate at which the volume of the cell culture medium being added (volume / unit of time) can be varied and depends on the conditions of the particular cell culture system. Culturing, and maintaining, the working volume typically occurs at the start of the cell culture before feed-up, or increasing the working volume to the final working volume, and can last from 1 to 5 days (e.g., 1 day, 2 days, 3 days, 4 days, or 5 days).

[0057] During the perfusion process, the volume can be changed by modulating the volume of spent cell culture media removed and volume of fresh cell culture media added. In the methods described herein, a feed-up, or increasing the working volume to the final working volume, is employed after 1 to 5 days (e.g., after 1 day, after 2 days, after 3 days, after 4 days, or after 5 days) after inoculation. A feed-up is a stage where the working volume of the bioreactor is increased to the final working volume over a period of many hours. The production bioreactor process begins with inoculation at a working volume that is 50% to 95% of the final working volume. 1 to 5 days into the culture, the working volume is gradually increased over a 12 to 96 hour, a 24 to 72 hour, or 36 to 48 hour period to the final working volume while spent media is removed at a specific flow rate. The feed media is at a specific flow that is greater than the spent media rate in order to increase the cell culture volume.

[0058] In one sub-embodiment, one or more feed rates are selected such that the feed up phase, or increasing the working volume to the final working volume, is maintained for at least 6 hours to 72 hours. In one sub-embodiment, the feed up phase, or increasing the working volume to the final working volume, is maintained for at least about 16 hours to at least about 40 hours or 30 hours ± 24 hours, or 30 hours ± 8 hours. In one sub-embodiment, the feed-up phase is maintained for at least 6, 10, 12, 16, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 48, 50, 54, or 60 hours.

[0059] During the feed-up phase, or increasing the working volume to the final working volume step, the feed and permeate rates are each independently maintained at rates less than or equal to 0.5 culture volumes / day (V / d), with the feed rate being greater than the permeate rate. In one subembodiment, the feed rate or the permeate rate is at least about 0.05 V / d to 0.50 V / d, at least about 0.05 V / d to at least about 0.45 V / d, at least about 0.05 V / d to at least about 0.40 V / d, at least about0.05 V / d to at least about 0.35 V / d, at least about 0.05 V / d to at least about 0.30 V / d, at least about0.05 V / d to at least about 0.25 V / d, at least about 0.05 V / d to at least about 0.20 V / d, at least about0.05 V / d to at least about 0.15 V / d, or at least about 0.05 V / d to at least about 0.10 V / d.

[0060] Similarly, the perfusion rate prior to and after feed-up, or increasing the working volume to the final working volume, can be less than or equal to 0.5 culture volumes / day (V / d), at least about 0.05 V / d, at least about 0.05 V / d to less than about 0.5 V / d, at least about 0.05 V / d to at least about 0.45 V / d, at least about 0.05 V / d to at least about 0.40 V / d, at least about 0.05 V / d to at least about 0.35 V / d, at least about 0.05 V / d to at least about 0.30 V / d, at least about 0.05 V / d to at least about 0.25 V / d, at least about 0.05 V / d to at least about 0.20 V / d, at least about 0.05 V / d to at least about 0.15 V / d, at least about 0.05 V / d to at least about 0.10 V / d, or at least about 0.1 V / d to about 0.5 V / d.

[0061] In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume, is initiated when the culture achieves one or more desired target criteria, including a desired culture volume, final culture volume, bioreactor working volume, final bioreactor working volume, time point, titer, cell density, packed cell volume, product attribute, process, production and / or a plant schedule.

[0062] In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is initiated when the culture reaches a desired cell density. In one subembodiment, the feed up phase is initiated when the cell density is at least 100 x 105cells / mL or up to350 x 105cells / mL. In one sub-embodiment, the cell density is at least about 100 x 105to about 350 x 105cells / mL, at least about 100 x 105to 300 x 105cells / mL, at least about 100 x 105to 250 x 105cells / mL, at least about 100 x 105to 200 x 105cells / mL, or at least about 100 x 105to 150 x 105cells / mL. In one sub-embodiment, the cell density is at least about 100 x 105, 125 x 105, 150 x 105, 175 x 105, 200 x 105, 225 x 105, 250 x 105, 275 x 105, 300 x 105, 325 x 105, or 350 x 105cells / mL.

[0063] In one sub-embodiment, the feed up phase, or increasing the working volume to the final working volume step, is initiated when the culture reaches a cell density in the range of about 100 x 105cells / mL to about 350 x 105cells / mL (e.g., about 100 x 105, about 125 x 105, about 150 x 105, about 175 x 105, about 200 x 105, about 225 x 105, about 250 x 105, about 275 x 105, about 300 x 105, about 325 x 105, or about 350 x 105cells / mL).

[0064] In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is initiated at least about 24 hours post-inoculation. In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is initiated up to 96 hours post-inoculation. In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is initiated about 24 hours to about 96 hours postinoculation. In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is initiated about 24 hours to about 72 hours post-inoculation. In one subembodiment, the feed-up phase, or increasing the working volume to the final working volume step, is initiated about 24 hours to about 48 hours post-inoculation. In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is initiated about 48 hours to about 96 hours post-inoculation. In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is initiated about 48 hours to about 72 hours postinoculation. In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is initiated about 72 hours to about 96 hours post-inoculation. In one subembodiment, the feed up phase, or increasing the working volume to the final working volume step, is initiated about 24, 48, 72, or 96 hours post-inoculation.

[0065] The feed-up phase, or increasing the working volume to the final working volume step, can be maintained until one or more desired target criteria is met. Such target criteria include, but are not limited to, a desired culture volume, a final culture volume, a desired bioreactor working volume,a final bioreactor working volume, time point, titer, cell density, packed cell volume, product attribute, process, production and / or a plant schedule.

[0066] In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is maintained for a desired length of time. In one sub-embodiment, the desired time is based on a process, production and / or plant schedule. In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is maintained for such a time as to allow for at least one population doubling of the culture. In one embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is maintained for a sufficient time such that the culture is not shocked due to the rapid increase in media concentrates.

[0067] In one sub-embodiment, the feed-up phase, or increasing the working volume to the final working volume step, is maintained until a desired culture volume is achieved. Generally, after feed- up, or increasing the working volume to the final working volume step, the desired culture volume is equal to the final working volume of the bioreactor. However, the feed-up, or increasing the culture volume to the final working volume, can be run until the culture volume is about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, of the final working volume of the bioreactor. The feed-up, or increasing the working volume to the final working volume step, can also be run until the culture volume is greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90% of the final working volume of the bioreactor. When the feed-up, or increasing the working volume to the final working volume step, does not result in the final working volume, a net addition of media can be performed to bring the cell culture to the final working volume. Thus, the feed-up, or increasing the working volume to the final working volume can occur in a step-wise manner to obtain the final working volume.

[0068] Once feed-up, or increasing the working volume to the final working volume step, has been completed, level control at the final working volume is enabled. In other words, the effective working volume in the bioreactor is maintained to a target setpoint. The permeate will “pull” culture out at a defined rate, the media feeds will then kick in to supplement that final working volume to maintain the cell culture at the final working volume. The complete cell culture production process or a production run is typically concluded at between 10 to 40 days, 10 and 20 days, preferably between 14 and 16 days.

[0069] The antifoam may be present in the cell culture medium in the bioreactor and / or can be added to the cell culture medium (either to the cell culture medium in the bioreactor or to the fresh perfusion medium being added to the bioreactor system) prior to the first culturing step (i.e. step a) and / or is added to the cell culture medium during the first culturing step. In certain embodiments, the level of antifoam at the start of feed-up, or increasing the working volume to the final working volume step, is greater than or equal to 22 mg / kg, greater than or equal to 24 mg / kg, greater than or equal to 26 mg / kg, greater than equal to 28 mg / kg, greater than or equal to 30 mg / kg, greater than or equal to 32 mg / kg, greater than equal to 34 mg / kg, greater than equal to 36 mg / kg, greater than equal to 38 mg / kg, or greater than equal to 40 mg / kg of cell culture.

[0070] The antifoams used in the methods disclosed herein are non-ionic silicone -based emulsions. An example is simethicone. Simethicone may be provided as a 1% solution, 2% solution, 5% solution, 10% solution or any concentration commercially available or any concentration capable of being formulated by one of skill in the art. When the antifoam is provided as containing a certain percentage of simethicone, it is the active ingredient which is calculated for the purposes of the disclosure. For example, when simethicone is provided as a 1% solution, the amount of antifoam in the bioreactor is based on the weight of simethicone, not the weight of the complete solution. Antifoams based on simethicone are also commercially available as, for example EX-CELL® antifoam (Sigma- Aldrich) and HyClone™ ADCF™ Antifoam agent (Cytiva).

[0071] In some embodiments, the method includes further feeding of antifoam into the bioreactor after feed-up or the cell culture volume has been increased to the final working volume. This can be to maintain the amount of antifoam and / or replenish the antifoam in the cell culture after feed-up or increasing the cell culture volume to the final working volume. In certain sub-embodiments of any of the methods described herein, the method includes increasing the antifoam in the cell culture to greater than or equal to 22 mg / kg, greater than or equal to 24 mg / kg, greater than or equal to 26 mg / kg, greater than equal to 28 mg / kg, greater than or equal to 30 mg / kg, greater than or equal to 32 mg / kg, greater than equal to 34 mg / kg, greater than equal to 36 mg / kg, greater than equal to 38 mg / kg, or greater than equal to 40 mg / kg of cell culture.

[0072] In certain embodiments, antifoam can be added after feed-up, or increasing the working volume to the final working volume, in an amount sufficient to maintain less than 50%, less than40%, less than 30%, less than 20%, or less than 10% foam coverage at the fluid surface of the bioreactor.

[0073] As the antifoam is being continuously supplied to the cell culture, in some subembodiments, the amount of antifoam at the end of the culturing is between 70 and 300 mg / kg.

[0074] The addition of antifoam in cell culture in perfusion culturing can be performed continuously, semi -continuously or as a bolus. In a continuous mode, the antifoam is added to the cell culture as part of its manufacture. In such a continuous mode, the rate of addition of antifoam is directly related to the rate of addition of perfusion media. In another continuous mode, antifoam could be fed continuously apart from the fresh culture media at any rate sufficient to provide the desired amount of antifoam. In a semi-continuous mode, a fixed bolus amount of antifoam is delivered to the bioreactor at a predefined frequency. A bolus adds a single amount of antifoam to the bioreactor. In certain embodiments, the antifoam is added to the bioreactor separately from the perfusion culture media. For example, a 1% simethicone solution can be added to achieve the desired amount of antifoam to reduce foam.

[0075] In some embodiments of any of the methods described herein, the bioreactor is a perfusion bioreactor. In some embodiments of any of the methods described herein, the perfusion bioreactor has a volume between about 1.5 L to about 25,000 L, or 200L to about 20,000L.

[0076] In some sub-embodiments of any of the methods described herein, the culture medium is selected from the group of a chemically-defined liquid culture medium, a serum-free liquid culture medium, a serum-containing liquid culture medium, an animal-derived component free liquid culture medium, and a protein-free medium.

[0077] In some sub-embodiments of any of the methods described herein, the mammalian cell is a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, an NSO cell, a baby hamster kidney (BHK) cell, a PerC6 cell, a Vero cell, or a HT-1080 cell line. In some subembodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. In some sub-embodiments, the mammalian cell is a human embryonic kidney (HEK) cell. In some sub-embodiments, the mammalian cell is an NSO cell. In some sub-embodiments, the mammalian cell is a baby hamster kidney (BHK) cell. In some sub-embodiments, the mammalian cell is a PerC6 cell. In some sub-embodiments, the mammalian cell is a Vero cell. In some sub-embodiments, the mammalian cell is a HT-1080 cell line.

[0078] In some sub-embodiments of any of the methods described herein, the recombinant protein is an immunoglobulin, an enzyme, a growth factor, a protein fragment, or an engineered protein. In some sub-embodiments, the recombinant protein is an antibody.DEFINITIONS

[0079] While the terminology used in this application is standard within the art, definitions of certain terms are provided herein to assure clarity and definiteness in the meaning of the claims. Units, prefixes, and symbols may be denoted in their SI (International System of Units) accepted form. Numeric ranges recited herein are inclusive of the numbers defining the range and include and are supportive of each integer within the defined range. The methods and techniques described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Eaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).

[0080] As used herein, the terms “a” and “an” mean one or more unless specifically indicated otherwise. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0081] All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference. What is described in an embodiment, sub-embodiment, or aspect of the invention can be combined with other embodiments, sub-embodiments or aspects of the invention, either singly or multiply.

[0082] As used herein, the term “bioreactor” means any vessel useful for the growth of a cell culture. The cell cultures of the instant disclosure can be grown in a bioreactor, which can be selected based on the application of a protein of interest that is produced by cells growing in the bioreactor. A bioreactor can be of any size so long as it is useful for the culturing of cells; typically, a bioreactor is sized appropriate to the volume of cell culture being grown inside of it. Typically, a bioreactor will be at least 1 liter and may be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1500, 2000, 2,500, 5,000, 8,000, 10,000, 12,000, 15,000, 20,000, or 25,000 liters or more, or any volume in between. The internal conditions of the bioreactor, including, but not limited to pH and temperature, can be controlled during the culturing period. Those of ordinary skill in the art will be aware of, and will be able to select, suitable bioreactors for use in practicing the methods disclosed herein based on the relevant considerations.

[0083] As used herein, “bioreactor working volume” or “working volume” refers to the volume within the bioreactor in which the cell culture is operated. For stainless steel bioreactors, the working volume is typically up to about 90% of the bioreactor volume. Some cell culture operations, including the perfusion culture processes described herein, make use of different percentages of the available working volume over the duration of the culture. “Final working volume,” as used herein, refers to the greatest volume within bioreactor in which the cell culture is operated, typically the volume during the production phase. As used herein, “culture volume” refers to the volume of all culture components that are in the bioreactor and attached equipment and associated flow paths, including the culture medium, cells, cell debris, bubbles, and foam. The culture volume is expressed as a percentage of the final working volume. As used herein, “kg of cell culture” refers to the total of the cell mass, cell culture media, and other components within the bioreactor.

[0084] As used herein, the term “cell density” refers to the number of cells in a given volume of culture medium. “Viable cell density” refers to the number of live cells in a given volume of culture medium, as determined by standard viability assays (such as trypan blue dye exclusion method).

[0085] As used herein, the term “cell viability” means the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term also refers to that portion of cells which are alive at a particular time in relation to the total number of cells, living and dead, in the culture at that time.

[0086] As used herein, the term “packed cell volume” (PCV), also referred to as “percent packed cell volume” (%PCV), is the ratio of the volume occupied by the cells, to the total volume of cell culture, expressed as a percentage (see Stettler et al., 2006, Biotechnol Bioeng. Dec 20:95(6): 1228-33). Packed cell volume is a function of cell density and cell diameter; increases in packed cell volume could arise from increases in either cell density or cell diameter or both. Packed cell volume is a measure of the solid content in the cell culture. Solids are removed during harvest and downstream purification, ore solids mean more effort to separate the solid material from the desired product during harvest and downstream purification steps. Also, the desired product can become trapped in the solids and lost during the harvest process, resulting in a decreased product yield. Since host cells vary in size and cell cultures also contain dead and dying cells and other cellular debris, packed cell volume is a more accurate way to describe the solid content within a cell culture than cell density or viable cell density. For example, a 2000L culture having a cell density of 50 x 106cells / ml would have vastly different packed cell volumes depending on the size of the cells. In addition, some cells, when in a growth-arrested state, will increase in size, so the packed cell volume prior to growtharrest and post growth-arrest will likely be different, due to increase in biomass as a result to cell size increase.

[0087] As used herein, the term “titer” means the total amount of a polypeptide or protein of interest (which may be a naturally occurring or recombinant protein of interest) produced by a cell culture in a given amount of medium volume. Titer can be expressed in units of milligrams or micrograms of polypeptide or protein per milliliter (or other measure of volume) of medium. “Cumulative titer” is the titer produced by the cells during the course of the culture, and can be determined, for example, by measuring daily titers and using those values to calculate the cumulative titer.

[0088] As used herein, the terms “feed,” “feed sample” and “feed stream” refer to the solution that is delivered (e.g., continuously, as a batch) to a fdtration module to be fdtered. The feed that is delivered to a fdtration module for fdtration can be, for example, feed from a feed container (e.g., vessel, tank) external to the system, or retentate from a preceding fdtration module in the same system.

[0089] As used herein, “feed channel” refers to a conduit for conveying a feed from a feed source (e.g., a feed container) to one or more processing units in a fdtration assembly.

[0090] As used herein, the term “filtration” generally refers to the act of separating the feed sample into two streams, a permeate and a retentate, using membranes.

[0091] As used herein, the terms “filtration membrane”, “membrane filter” or “filter” refers to a selectively permeable membrane for separating a feed into a permeate stream and a retentate stream using a TFF process. Filtration membranes include, but are not limited to, ultrafiltration (UF) membranes, microfiltration (MF) membranes, reverse osmosis (RO) membranes and nanofiltration (NF) membranes.

[0092] As used herein, the terms “permeate” and “filtrate” refer to that portion of the feed that has permeated through the membrane.

[0093] As used herein, “permeate channel” refers to a conduit in a filtration assembly for carrying permeate.

[0094] As used herein, the term “retentate” refers to the portion of the solution that has been retained by the membrane, and the retentate is the stream enriched in a retained species.

[0095] As used herein, “retentate channel” refers to a conduit in a filtration assembly for carrying retentate.

[0096] As used herein, the term “microfiltration membranes” and “MF membranes” are used herein to refer to membranes that have pore sizes in the range between about 0.1 micrometers to about 10 micrometers.

[0097] As used herein, the terms “ultrafiltration membrane” and “UF membrane” are used herein to refer to a membrane that has pore sizes in the range of between about 1 nanometer to about 100 nanometers.

[0098] As used herein, the term “flow path” refers to a channel supporting the flow of a liquid (e.g., feed, retentate, permeate) through all or part of a system. The flow path can have any topology which supports tangential flow (e.g., straight, coiled, arranged in zigzag fashion). The flow path can be parallel or serial. A flow path can also refer to a path resulting in a single pass through a system or a path for recirculating retentate through a TFF system. Furthermore, a flow path can be open, as in an example of channels formed by hollow fiber membranes.

[0099] As used herein, the term “fluidly connected” refers to two or more components of a TFF system that are connected by one or more conduits (e.g., a feed channel, a retentate channel, a permeate channel) such that a liquid can flow from one component to the other.

[0100] As used herein, the term “perfusion flow rate” or "perfusion rate" is the amount of media that is passed through (added and removed) from a bioreactor, typically expressed as some portion or multiple of the working volume, in a given time. In some embodiments, the term "perfusion rate" is used when the feed rate (the amount of media that is added to a bioreactor) is the same as the contemporaneous permeate rate (the amount of media that is removed from the bioreactor) and refers to that rate of addition / removal.

[0101] As used herein, the term “processing” refers to the act of filtering a feed containing a product of interest and subsequently recovering the product in a concentrated form. The concentrated product can be recovered from the filtration system in either the retentate stream or permeate stream depending on the product's size and the pore size of the filtration membrane.

[0102] As used herein, the term “product” refers to a target compound in a feed. Typically, a product will be a biomolecule (e.g., protein) of interest, such as a monoclonal antibody (mAb).FOAM COVERAGE

[0103] Maintaining foam coverage at a target of 10% or less of the fluid surface of the bioreactor during initial mammalian cell culturing helps prevent filter fouling when recirculating tangential flow (RTF) filtration systems are used in perfusion process that include a feed-up step. As demonstrated in the Examples, controlling foam buildup on the reactor surface prevents accumulation of cell debris on the bioreactor vessel sidewalls, which can otherwise form a "crust layer" that leads to catastrophic filter fouling when introduced into the working volume during feed-up.

[0104] In some embodiments, the foam coverage at the fluid surface of the bioreactor may be maintained at a target of 9% or less. In some embodiments, the foam coverage may be maintained at a target of 8.5% or less. In some embodiments, the foam coverage may be maintained at a target of 8% or less. In some embodiments, the foam coverage may be maintained at a target of 7.5% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor may be maintained at a target of 7% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor may be maintained at a target of 6.5% or less. In some embodiments, the foam coverage at the fluidsurface of the bioreactor may be maintained at a target of 6% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor may be maintained at a target of 5.5% or less. In some embodiments, the foam coverage may be maintained at a target of 5% or less. In some embodiments, the foam coverage may be maintained at a target of 4.5% or less. In some embodiments, the foam coverage may be maintained at a target of 4% or less. In some embodiments, the foam coverage may be maintained at a target of 3.5% or less. In some embodiments, the foam coverage may be maintained at a target of 3% or less. In some embodiments, the foam coverage may be maintained at a target of 2.5% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor may be maintained at a target of 2% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor may be maintained at a target of 1.5% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor may be maintained at a target of 1% or less. In some embodiments, the foam coverage at the fluid surface of the bioreactor may be maintained at a target of 0.5% or less.

[0105] If foam coverage exceeds the target coverage, antifoam (e.g., simethicone) can be added to quickly reduce foam levels. Rapid response to foam buildup can prevent extended periods of high foam coverage that could lead to problematic debris accumulation. In some embodiments, foam coverage may temporarily increase to ± 50% (e.g., ± 45%, ± 40%, ± 35%, ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5%) of the target coverage before being brought back down below the target through antifoam addition.

[0106] In some embodiments, if foam coverage exceeds the target coverage, antifoam may be delivered as one or more boluses to quickly reduce foam levels. The bolus amount may be predetermined or adjusted based on the extent of foam coverage. In some cases, a single bolus of antifoam may be sufficient to bring the foam coverage below the target level. In other instances, multiple smaller boluses may be administered sequentially until the desired foam reduction is achieved. In certain embodiments, the size of the antifoam bolus may be calculated based on the current working volume of the bioreactor.

[0107] In some embodiments, the antifoam may be delivered through a dedicated antifoam addition port in the bioreactor. Alternatively, the antifoam may be added directly to the cell culture medium. The delivery of antifoam boluses may be automated based on foam sensors or visual monitoring systems, or may be manually controlled by an operator.ANTIFOAM

[0108] Antifoam agents play a important role in upstream cell culture processes by preventing excessive foam formation, which can interfere with gas exchange, nutrient availability, and overall culture performance. In the cell culture methods described herein, the amount of antifoam present at the initiation of increasing the working volume to the final working volume (feed-up) is particularly important for maintaining optimal culture conditions and preventing filter fouling in recirculating tangential flow (RTF) filtration systems.

[0109] In some embodiments, the amount of antifoam in the bioreactor at the initiation of increasing the working volume to the final working volume is at least 14 mg antifoam / kg of cell culture (mg / kg), wherein the amount of antifoam is calculated based on the final working volume. In certain embodiments, the amount of antifoam at the initiation of increasing the working volume is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is at least 15 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is at least 16 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is at least 17 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is at least 18 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is at least 19 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is at least 20 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is at least 21 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is at least 22 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is at least 23 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is at least 24 mg / kg.

[0110] In some embodiments, the amount of antifoam at the initiation of increasing the working volume is no more than 37, 38, 39, 40, 45, 50, or 60 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is no more than 37 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is no more than 38 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is no more than 39 mg / kg. In some embodiments, the amount of antifoam at theinitiation of increasing the working volume is no more than 40 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is no more than 45 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is no more than 50 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is no more than 60 mg / kg.

[0111] In some embodiments, the amount of antifoam at the initiation of increasing the working volume is in the range of 15 mg / kg to 37 mg / kg, 15 mg / kg to 38 mg / kg, 15 mg / kg to 39 mg / kg, 15 mg / kg to 40 mg / kg, 15 mg / kg to 45 mg / kg, 15 mg / kg to 50 mg / kg, or 15 mg / kg to 60 mg / kg. In some embodiments, the amount of antifoam at the initiation of increasing the working volume is in the range of 16 mg / kg to 37 mg / kg, 16 mg / kg to 38 mg / kg, 16 mg / kg to 39 mg / kg, 16 mg / kg to 40 mg / kg, 16 mg / kg to 45 mg / kg, 16 mg / kg to 50 mg / kg, or 16 mg / kg to 60 mg / kg.

[0112] The antifoam used in the methods described herein may be a silicone-based antifoam. Silicone-based antifoams are effective at reducing surface tension and breaking foam bubbles in cell culture media. In some embodiments, the antifoam is simethicone.

[0113] In certain embodiments where simethicone is used as the antifoam agent, the amount of simethicone in the bioreactor at the initiation of increasing the working volume to the final working volume is at least 14 mg simethicone / kg of cell culture (mg / kg), wherein the amount of simethicone is calculated based on the final working volume.

[0114] In some embodiments, the amount of simethicone at the initiation of increasing the working volume is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is at least 15 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is at least 16 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is at least 17 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is at least 18 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is at least 19 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is at least 20 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is at least 21 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is at least 22 mg / kg. In some embodiments, the amount ofsimethicone at the initiation of increasing the working volume is at least 23 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is at least 24 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is in the range of 15 mg / kg to 37 mg / kg, 15 mg / kg to 38 mg / kg, 15 mg / kg to 39 mg / kg, 15 mg / kg to 40 mg / kg, 15 mg / kg to 45 mg / kg, 15 mg / kg to 50 mg / kg, or 15 mg / kg to 60 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is in the range of 15 mg / kg to 37 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is in the range of 15 mg / kg to 38 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is in the range of 15 mg / kg to 39 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is in the range of 15 mg / kg to 40 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is in the range of 15 mg / kg to 45 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is in the range of 15 mg / kg to 50 mg / kg. In some embodiments, the amount of simethicone at the initiation of increasing the working volume is in the range of 15 mg / kg to 60 mg / kg.

[0115] The antifoam agent may be delivered to the bioreactor using various methods. In some embodiments, the antifoam is added in a continuous or semi-continuous manner. In other embodiments, the antifoam is added as a single bolus. The antifoam may be added to the bioreactor separately from the perfusion culture media. In some embodiments, simethicone is added via a 1% simethicone solution. The method of antifoam delivery can be selected based on the specific requirements of the cell culture process and the desired foam control strategy.PERFUSION CULTURE

[0116] In the methods described herein, mammalian cells expressing the recombinant protein are cultured under perfusion conditions. Such conditions are well known to those skilled in the art and are described in detail below. Perfusion culture, sometimes known as continuous culture, is one in which the cell culture receives the addition of fresh medium (“perfusion medium”) and spent medium is removed from the bioreactor. Perfusion can be continuous, step-wise, intermittent, or a combination of any or all of any of these. Perfusion rates can be less than a working volume to many working volumes per day. The perfusion flow rate can also be one working volume or less per day. Perfusionfeed medium can be formulated to maximize perfusion nutrient concentration to minimize perfusion rate.

[0117] The perfusing can be by continuous perfusion. The rate of perfusion can be constant. In one example, the perfusing is performed at a rate of less than or equal to 1.0 working volume per day. In one example, the perfusing is accomplished by a peristaltic pump, a double diaphragm pump, a low shear pump or alternating tangential flow.

[0118] Preferably the cells are retained in the culture and the spent medium that is removed is substantially free of cells or has significantly fewer cells than the cell culture. Recombinant proteins expressed by the cell culture may be retained or removed from the cell culture, depending on the retention system used. Sometimes it is preferable for the host cells and the expressed recombinant proteins to remain in the retentate in the bioreactor and for the permeate to be substantially free of or have significantly less of either (“null permeate”). Other times it may be preferable to retain cells but allow the expressed proteins to pass into the permeate (“harvest permeate”).

[0119] Perfusion can be accomplished by a number of means including centrifugation, sedimentation, or filtration. See e.g. Voisard et al., (2003), Biotechnology and Bioengineering 82:751- 65. In one embodiment, a filtration method is used. Filters include membrane filters, ceramic filters and metal filters and may be in any shape, including spiral wound or tubular or in the form of a flat sheet. One or more filters can be connected to, in fluid communication with, a bioreactor together or independently, in series or in parallel. In certain embodiments, a hollow-fiber filter is used.

[0120] Some sub-embodiments of any of the methods described herein further include subjecting the permeate to microfiltration and harvesting the cells to collect recombinant protein from the mammalian cells. Some sub-embodiments of any of the methods described herein further include formulating the collected recombinant protein into a pharmaceutical composition.

[0121] In some sub-embodiments of any of the methods described herein, collecting (i.e., harvesting) is performed after the culturing is performed for a total of 10, 12, 14, 16, 18 or 20 days. The cells may be harvested and chilled for subsequent processing.

[0122] The rate of perfusion can be any rate appropriate to the cell culture. The perfusion rate has no impact on inhibiting foam tolerance. Runs can be performed at any rate of the working volume and any rate of the final working volume. For example, the rate of perfusion can range from about 0.1vvd to about 20 wd, or more preferably from about 0.5 vvd to about 10 vvd , or most preferably from about 0.5 vvd to about 2.5 vvd. The rate of perfusion can remain constant over a period of time, or can be altered (i.e., increased or decreased) over the course of a period of perfusion, or any combination thereof. Further, an increase or decrease in the rate of perfusion can be applied in any manner known in the art, including, but not limited to, a steady alteration over time, e.g., a steady increase during a period of perfusion, or a series of alterations over time, e.g., a series of steady alterations, a series of stepwise alterations (e.g., the rate of perfusion could be increased or decreased in a stepwise manner), or any combination thereof. The perfusion can be applied in a continuous manner or in an intermittent manner, as noted above. The timing of the initiation and cessation of a perfusion period (s), and of any alterations to the perfusion, can be predetermined, e.g., at a set time (s) or interval (s), or based upon the monitoring of some parameter or criterion.NON-LIMITING EXAMPLE PERFUSION CULTURE METHODS

[0123] Initiating a perfusion cell culture at a culture volume less than the final working volume and then feeding up to the final working volume allows for efficient use of the bioreactor during both growth and production phases. This approach, which is described in International Patent Application Publication No. WO2024 / 054414, enables high cell densities to be achieved quickly in a reduced volume during the growth phase, while the subsequent increase to the final working volume provides improved conditions for protein production. Perfusion methods using this type of feed-up strategy reduce the amount of feed media required and waste generated compared to traditional perfusion cultures maintained at the final working volume throughout the growth and production phases. RTF filtration is particularly well-suited for these intensified perfusion processes due to its reduced prep time, improved scalability, and more flexible facility fit relative to ATF filtration. However, RTF systems are more susceptible to filter clogging during feed-up than ATF systems because their unidirectional flow may not effectively clear debris that accumulates at the filter inlet, whereas ATF's bidirectional flow pattern may better dislodge such material. The use of specific amounts of antifoam, particularly prior to feed-up, helps prevent the accumulation of cellular debris at the fluid surface and subsequent filter inlet clogging that can occur with RTF systems, thereby addressing a key challenge of RTF filtration while still leveraging its advantages for intensified processes.

[0124] The present disclosure provides methods for producing a recombinant protein, preferably an antibody, comprising: a) culturing mammalian cells expressing a recombinant protein in abioreactor under perfusion and recirculating tangential flow (RTF) fdtration conditions sufficient to produce the recombinant protein and at a working volume from 50% to 95%, preferably 50% to 75%, of the final working volume; b) maintaining the foam coverage at the fluid surface of the bioreactor at a target of 10% or less through the presence of antifoam; c) increasing the working volume to the final working volume, wherein the cell culture within the bioreactor contains antifoam in an amount of at least 14 mg antifoam / kg of cell culture (mg / kg), preferably in the range of 23 mg / kg to 38 mg / kg, at the initiation of increasing the working volume, wherein the amount of antifoam is calculated based on the final working volume; and d) culturing the mammalian cells for the duration of a production run. In some embodiments, the amount of antifoam at step c) may be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 mg / kg. The method may include culturing in step a) with a bioreactor inoculated with 1 x 106to 20 x 106cells / ml, and in some cases, the increasing the working volume step may be initiated between 24 to 120 hours after initiation of the culturing in step a). The antifoam may be added in a continuous or semi-continuous manner or as a bolus, and in certain preferred embodiments, the antifoam is simethicone. In some preferred embodiments, the amount of antifoam when the culture is terminated or harvested is between 90 mg / kg and 220 mg / kg. In some embodiments, the amount of antifoam when the culture is terminated or harvested is between 100 mg / kg and 200 mg / kg. In some embodiments, the amount of antifoam when the culture is terminated or harvested is between 110 mg / kg and 180 mg / kg.

[0125] The present disclosure further provides methods for producing a recombinant protein, preferably an antibody, comprising: initiating a culture in a bioreactor at a working volume that is at least 50% of a final working volume, preferably 50% to 75% of the final working volume; inoculating the culture with mammalian cells, preferably CHO cells, engineered to express the recombinant protein; perfusing the culture at one or more perfusion rates of less than or equal to 0.5 culture volumes / day (V / d) until the culture reaches one or more desired target criteria, wherein foam coverage at the fluid surface of the bioreactor is maintained at 10% or less during the perfusing; increasing the working volume to the final working volume, wherein the bioreactor contains antifoam, preferably simethicone, in an amount of at least 14 mg antifoam / kg of cell culture (mg / kg), preferably in the range of 23 mg / kg to 38 mg / kg, at the initiation of increasing the working volume, wherein the amount of antifoam is calculated based on the final working volume; and once the final working volume is achieved, perfusing the culture at one or more perfusion rates of less than or equal to 0.5V / d until the culture is terminated or harvested, wherein all rates expressed in V / d are calculated based on the final working volume.

[0126] In some embodiments, the culture is initiated at a working volume that is 50% to 75% of the final working volume. In some embodiments, the culture is initiated at a working volume that is 60% to 70% of the final working volume. In some embodiments, the culture is initiated at a working volume that is 65% to 75% of the final working volume.

[0127] In some embodiments, the culture is inoculated at a cell density of 5 x 106cells / mL to 50 x 106cells / mL. In some embodiments, the culture is inoculated at a cell density of 6 x 106cells / mL to 20 x 106cells / mL. In some embodiments, the culture is inoculated at a cell density of 8 x 106cells / mL to 12 x 106cells / mL.

[0128] In some embodiments, the culture may be maintained in batch mode for a period of time following inoculation. During this initial batch culture, no media is added to or removed from the culture. The duration of the batch mode may vary depending on the specific cell line and culture conditions. In some cases, batch mode may be maintained for up to 48 hours post-inoculation. In other embodiments, batch mode may be maintained for about 12 to 24 hours, or about 24 to 36 hours. The length of the initial batch culture may be optimized based on factors such as cell growth rate, metabolite production, and overall process goals. In some embodiments, batch mode is maintained for less than or equal to 24 hours post-inoculation. In some embodiments, batch mode is maintained for about 24 hours post-inoculation. Following the batch culture, perfusion may be initiated through the continuous addition of fresh media and removal of spent media.

[0129] In some embodiments, the culture is perfused at one or more perfusion rates of 0.05 V / d to 0.5 V / d until the culture reaches one or more desired target criteria. In some embodiments, the culture is perfused at one or more perfusion rates of 0. 10 V / d to 0.25 V / d until the culture reaches one or more desired target criteria. In some embodiments, the culture is perfused at one or more perfusion rates of 0.13 V / d until the culture reaches one or more desired target criteria.

[0130] In some embodiments, the desired target criteria for increasing the culture volume to the final working volume may include reaching a specific cell density, such as 100 x 105cells / mL to 350 x 105cells / mL. In some embodiments, the desired target criteria may be based on a time point postinoculation, such as 24 hours to 72 hours after initiating the culture. In some embodiments, the desiredtarget criteria may include achieving a certain titer level or packed cell volume. In some embodiments, the desired target criteria may be related to specific product attributes or process parameters. In some embodiments, the desired target criteria may be determined based on production schedules or plant logistics. In some embodiments, a combination of two or more of these criteria may be used to determine when to initiate the increase in culture volume to the final working volume.

[0131] In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 9% or less during the perfusing. In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 8% or less during the perfusing. In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 7.5% or less during the perfusing. In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 5% or less during the perfusing. In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 2.5% or less during the perfusing. In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 1% or less during the perfusing.

[0132] In some embodiments, the culture volume is increased to the final working volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates, wherein at least one of the one or more feed rates is less than or equal to 0.40 V / d and at least one of the one or more permeate rates is less than or equal to 0.15 V / d. In some embodiments, the culture volume is increased to the final working volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates, wherein at least one of the one or more feed rates is less than or equal to 0.35 V / d and at least one of the one or more permeate rates is less than or equal to 0.10 V / d. In some embodiments, the culture volume is increased to the final working volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates, wherein at least one of the one or more feed rates is less than or equal to 0.30 V / d and at least one of the one or more permeate rates is less than or equal to 0.05 V / d.

[0133] In some embodiments, once the final working volume is achieved, the culture is perfused at one or more perfusion rates of 0.05 V / d to 0.5 V / d until the culture is terminated or harvested, wherein the feed rate(s) and the contemporaneous permeate rate(s) are the same. In some embodiments, once the final working volume is achieved, the culture is perfused at one or more perfusion rates of 0. 10 V / d to 0.25 V / d until the culture is terminated or harvested. In someembodiments, once the final working volume is achieved, the culture is perfused at one or more perfusion rates of 0.22 V / d until the culture is terminated or harvested.

[0134] In some embodiments, the amount of antifoam when the culture is terminated or harvested is between 90 mg / kg and 220 mg / kg. In some embodiments, the amount of antifoam when the culture is terminated or harvested is between 100 mg / kg and 200 mg / kg. In some embodiments, the amount of antifoam when the culture is terminated or harvested is between 110 mg / kg and 180 mg / kg.

[0135] The present disclosure also provides methods for producing a recombinant protein, preferably an antibody, comprising: initiating a culture in a bioreactor at a working volume that is at least 50% of a final working volume, preferably 50% to 75% of the final working volume; inoculating the culture with mammalian cells, preferably CHO cells, engineered to express the recombinant protein; perfusing the culture for 24 hours to 72 hours at one or more perfusion rates of 0.05 V / d to 0.5 V / d, preferably 0.10 V / d to 0.25 V / d, wherein foam coverage at the fluid surface of the bioreactor is maintained at 10% or less during the perfusing; increasing the working volume to the final working volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates, wherein at least one of the one or more feed rates is less than or equal to 0.50 V / d and at least one of the one or more permeate rates is less than or equal to 0.20 V / d, wherein each of the one or more feed rates is greater than the contemporaneous permeate rate and further wherein the bioreactor contains simethicone in an amount of at least 14 mg simethicone / kg of cell culture (mg / kg) at the initiation of increasing the working volume, wherein the amount of simethicone is calculated based on the final working volume; and once the final working volume is achieved, perfusing the culture at one or more perfusion rates of less than or equal to 0.5 V / d until the culture is terminated or harvested, wherein the amount of simethicone when the culture is terminated or harvested is between 80 mg / kg and 240 mg / kg, preferably between 100 mg / kg and 150 mg / kg, wherein all rates expressed in V / d are calculated based on the final working volume.

[0136] In some embodiments, the culture is initiated at a working volume that is 50% to 75% of the final working volume. In some embodiments, the culture is initiated at a working volume that is 60% to 70% of the final working volume. In some embodiments, the culture is initiated at a working volume that is 65% to 75% of the final working volume.

[0137] In some embodiments, the culture is inoculated at a cell density of 5 x 106cells / mL to 50 x 106cells / mL. In some embodiments, the culture is inoculated at a cell density of 6 x 106cells / mL to 20 x 106cells / mL. In some embodiments, the culture is inoculated at a cell density of 8 x 106cells / mL to 12 x 106cells / mL.

[0138] In some embodiments, the culture may be maintained in batch mode for a period of time following inoculation. During this initial batch culture, no media is added to or removed from the culture. The duration of the batch mode may vary depending on the specific cell line and culture conditions. In some cases, batch mode may be maintained for up to 48 hours post-inoculation. In other embodiments, batch mode may be maintained for about 12 to 24 hours, or about 24 to 36 hours. The length of the initial batch culture may be optimized based on factors such as cell growth rate, metabolite production, and overall process goals. In some embodiments, batch mode is maintained for less than or equal to 24 hours post-inoculation. In some embodiments, batch mode is maintained for about 24 hours post-inoculation. Following the batch culture, perfusion may be initiated through the continuous addition of fresh media and removal of spent media.

[0139] In some embodiments, the culture is perfused at one or more perfusion rates of 0.05 V / d to 0.5 V / d until feed-up. In some embodiments, the culture is perfused at one or more perfusion rates of 0.10 V / d to 0.25 V / d until feed-up. In some embodiments, the culture is perfused at one or more perfusion rates of 0. 13 V / d until feed-up.

[0140] In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 9% or less during the perfusing. In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 8% or less during the perfusing. In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 7.5% or less during the perfusing. In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 5% or less during the perfusing. In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 2.5% or less during the perfusing. In some embodiments, foam coverage at the fluid surface of the bioreactor is maintained at 1% or less during the perfusing.

[0141] In some embodiments, the culture volume is increased to the final working volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates, wherein at least one of the one or more feed rates is less than or equal to 0.40 V / d and at least one of the one or more permeate rates is less than or equal to 0.15 V / d. In someembodiments, the culture volume is increased to the final working volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates, wherein at least one of the one or more feed rates is less than or equal to 0.35 V / d and at least one of the one or more permeate rates is less than or equal to 0.10 V / d. In some embodiments, the culture volume is increased to the final working volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates, wherein at least one of the one or more feed rates is less than or equal to 0.30 V / d and at least one of the one or more permeate rates is less than or equal to 0.05 V / d.

[0142] In some embodiments, once the final working volume is achieved, the culture is perfused at one or more perfusion rates of 0.05 V / d to 0.5 V / d until the culture is terminated or harvested, wherein the feed rate(s) and the contemporaneous permeate rate(s) are the same. In some embodiments, once the final working volume is achieved, the culture is perfused at one or more perfusion rates of 0. 10 V / d to 0.25 V / d until the culture is terminated or harvested. In some embodiments, once the final working volume is achieved, the culture is perfused at one or more perfusion rates of 0.22 V / d until the culture is terminated or harvested.

[0143] In some embodiments, the amount of antifoam when the culture is terminated or harvested is between 90 mg / kg and 220 mg / kg. In some embodiments, the amount of antifoam when the culture is terminated or harvested is between 100 mg / kg and 200 mg / kg. In some embodiments, the amount of antifoam when the culture is terminated or harvested is between 110 mg / kg and 180 mg / kg.FILTRATION

[0144] Tangential flow filtration (TFF) is a separation process that uses membranes to separate components in a liquid solution or suspension on the basis of size, molecular weight or other differences. TFF is used in perfusion processes to remove target proteins from cell culture media, while retaining cells within the media. In TFF processes, fluid is pumped tangentially along the membrane surface and particles, molecules, or cells that are too large to pass through the membrane are rejected and returned to a process tank. TFF processes can involve additional passes of the fluid across the membrane (e.g., recirculation) until the process fluid is sufficiently clarified, concentratedor purified. The cross-flow nature of TFF minimizes membrane fouling, thus permitting high volume processing per batch.

[0145] In certain embodiments the filter unit comprises a microfiltration membrane and the permeate comprises a recombinant protein expressed by the cells. This allows for continuous harvest of the recombinant protein, which can be e.g. a protein such as an immunoglobulin.

[0146] In some embodiments the filter unit comprises an ultrafiltration membrane and a recombinant protein expressed by the cells is retained in the retentate and returned to the bioreactor. This allows for continuous removal of toxic or otherwise undesirable low molecular weight waste products / metabolites from the cell culture, while the recombinant protein, e.g. a protein such as an immunoglobulin, can be harvested from the culture at the end of the cultivation.

[0147] In certain embodiments, the permeate is collected from a filter that is a hollow fiber filter, a flat sheet membrane filter or a spiral wound membrane filter having a pore size or molecular weight cut off that does not retain the recombinant protein in the bioreactor.

[0148] In one sub-embodiment, the filter employed is a single unit filter system. In a related subembodiment, the single unit filter system comprises at least one hollow fiber filter component having a pore size or molecular weight cut off (MWCO) that retains the recombinant protein in the bioreactor and at least one hollow fiber filter component having a pore size or molecular weight cut off (MWCO) that does not retain the recombinant protein in the bioreactor. In a related sub-embodiment the molecular weight cutoff of at least one hollow fiber filter component that retains the recombinant protein in the bioreactor is 300 kDa or less. In a related sub-embodiment, the molecular weight cutoff of at least one hollow fiber filter component that does not retain the recombinant protein in the bioreactor is at least 500 kDa.

[0149] In one sub-embodiment, the at least one hollow fiber filter component that retains the recombinant protein in the bioreactor is an ultrafilter and at least one hollow fiber filter component that does not retain the recombinant protein in the bioreactor is a microfilter. In a related subembodiment the single unit filter system is contained within a housing. In a related sub-embodiment the single unit filter system further comprises a spacer between at least two of the hollow fiber filter components.

[0150] Hollow fiber filters are used in mammalian cell perfusion culture for cell and / or recombinant protein product retention. When the cell culture, including cell culture media, cells (whole and lysed), soluble expressed recombinant proteins, host cell proteins, waste products and the like, are introduced to the filter, depending on the pore size or molecular weight cutoff (MWCO) the hollow fiber material may retain certain cell culture components on the lumen side (inside) and allow certain components to pass through the filter (permeate) based on the pore size or molecular weight cutoff of the hollow fiber material. The material that is retained (retentate) is returned to the bioreactor. Fresh perfusion cell culture media is added to the bioreactor and permeate is withdrawn from the filter at predetermined intervals or continuously to maintain a desired or constant bioreactor volume. The permeate can be discarded, stored in holding tanks, bags or totes or transferred directly to another unit operation, such as filtration, centrifugation and / or other downstream purification methods or the like. Hollow fibers for microfiltration typically have a pore size ranging from 0. 1 pm to 5-10 pm or a molecular weight cut off of 500 kDa or more and can be used to allow the protein to pass through into the permeate.

[0151] For the methods described herein, employing a hollow fiber filter, the hollow fiber filter is generally made as a cartridge that comprises multiple hollow fibers (HF) that run, in parallel, the length of the cartridge and are embedded at each end of the cartridge (preferably with a potting agent); the lumens at the end of the HFs are retained open, thus forming a continuous passage through each of the lumens from one end of the cartridge to the other, i.e., from a cartridge entrance end, to a cartridge exit end. The hollow fibers are enclosed by the outer wall of the cartridge (i.e., the cartridge wall) and a potting layer at their ends. As a result, there is a chamber bounded by the cartridge wall and the outer walls of the HFs. That chamber can be used as the filtrate chamber. The intraluminar (internal) spaces of the HFs are considered collectively to constitute part of the retentate chamber in each of the present systems.

[0152] Ultrafiltration hollow fibers typically have a pore size range of 0.01 pm to 0. 1 pm or a molecular weight cut off of 300 kDa or less, and can be used to retain the desired protein in the retentate and return it back to the bioreactor. This can be used, for example, to concentrate the recombinant protein product for harvest. Such filters are available commercially, such as Xampler UFP-750-E-4MA, Xampler UFP-30-E-4MA, (GE Healthcare, Pittsburgh, Pa.) and Midikros TC Modules T02-E030-10, T02-050-10, T02- E750-05, T02-M10U-06 (Spectrum Laboratories, Inc,Dominguez, Calif.). In one embodiment, the filter is an MF filter which has a MWCO of 750 kDa and / or a pore size of 0.2 mm.

[0153] The pore size of the filter determines which substances will pass through to the permeate and which substances will be retained. In one embodiment of the invention, the MF device has 0.2 micron pore size. In another embodiment of the invention, the UF device has a pore size that allows only proteins smaller than 50,000 Daltons to pass through to the permeate. Thus, one skilled in the art will recognize that using a UF device with a 50,000 Dalton pore size will retain 100 % or nearly 100 % of the polypeptide product in a large - scale cell culture designed for antibody production (because the molecular weight of an antibody is typically about 150,000 Daltons). One skilled in art will also recognize that the pore size of the fdter can be varied depending on the size of the final polypeptide product (e.g., in order to retain the optimal amount of the final polypeptide product) or the size of the waste product to be removed.RTF SYSTEMS

[0154] In the methods disclosed herein, mammalian cells expressing the recombinant protein are cultured under recirculating tangential flow (RTF) conditions. RTF conditions are known to those skilled in the art and are described in more detail below. In addition to the bioreactor and filters described above, RTF systems employ pumps, most typically a reciprocating pump. The reciprocating pump can suitably comprise a reciprocating moving member, such as e.g. a diaphragm, a membrane or a piston. The reciprocating moving member can move back and forth in relation to a pump chamber (also called a cylinder, when the moving member is a piston), forcing fluid (e.g. culture liquid) out from the pump chamber during an inward stroke of the moving member and sucking fluid (e.g. culture liquid) into the pump chamber during an outward stroke. The stroke volume of the reciprocating pump corresponds to the fluid (culture liquid). Volume displaced out from or into the pump chamber during each stroke.

[0155] The reciprocating pump may e.g. be a fluid driven diaphragm pump, with a pump chamber and a drive fluid-filled drive chamber separated by a flexible diaphragm, which constitutes the reciprocating moving member. The drive fluid can be a gas, e.g. air, or a liquid. When fluid pressure is applied to the drive chamber via a drive fluid supply line, the diaphragm expels liquid from the pump chamber in an inward stroke and when the fluid pressure is released, the diaphragmflexes back and draws liquid into the pump chamber in an outward stroke. The pump chamber may e.g. be directly connected to the retentate inlet compartment of the filter unit at a joint. Alternatively, it may be connected via a fluid connector. Such as a short piece of tubing with a diameter large enough not to impede the liquid flow and a volume significantly smaller than the stroke volume of the reciprocating pump (e.g. less than 20% of the stroke volume, such as less than 10% or less than 5% of the stroke volume), optionally via an aseptic connector. Examples of suitable magnetic levitation pumps include Levitronix® Puralev® Series pump (Levitronix Technologies, Framingham, MA). Examples of suitable diaphragm pumps include Repligen XCell™ ATF pump (Repligen, Waltham, MA). Examples of suitable peristaltic pumps include Watson Marlow Series 500 and Series 600 pumps (Watson Marlow, Wilmington, MA).

[0156] The connections between the various components of the system can be achieved e.g. by tubing, such as flexible tubing, with the valves in-line with the tubing or adjacent to the branch point or the inlet / outlet ends. The branch point may be e.g. a three-way tubing connector or a manifold. The connection between the branch point and the bioreactor may be a single line in the form of a length of tubing, such as flexible tubing. The tubing length may be connected to the inner volume of the bioreactor vessel through a port in the vessel wall. The valves may be of any suitable check valve type, e.g. flap, ball, slit disk valves etc.

[0157] The construction materials used in the system can suitably be compatible with commonly used sterilization methods, such as e.g. gamma irradiation and / or autoclaving. For reusable components, stainless steel (e.g. with corrosion resistance at least equivalent to 316 L) or engineering plastics such as polysulfone, PEEK, etc. may be used, while for single-use components, plastics, such as e.g. polysulfone, polypropylene, polyethylene or ethylene copolymers, may be used.

[0158] Existing TFF devices used in perfusion systems include XCell™ ATF System (Repligen, Waltham, MA) and KrosFlo® Perfusion System (Spectrum Laboratories, Rancho Dominguez, CA), which are hollow fiber devices. These devices contain open feed channels, so as to limit physical damage to cells in the feed stream, and both devices require high cross-flow rates to minimize fouling {i.e., the accumulation of particles along the wall of membrane). Membrane fouling reduces product recovery because the passage of target proteins and waste materials through the membrane (i.e., sieving) is reduced. Eventually, membrane fouling can result in failure of the device, with product no longer being recovered during filtration. Custom TFF devices can also be used.

[0159] In certain sub-embodiments, the bioreactor comprises an inflatable flexible bag, resting on a movable support, and the length of tubing is a flexible length of tubing. The system of the invention is particularly suitable for bioreactors which are agitated by moving the entire bioreactor vessel (i.e. the inflatable flexible bag), since only one length of tubing is needed to connect to the bioreactor, which keeps the number of moving connections to the vessel low and reduces entangling of these.CELL CULTURE

[0160] As used herein, “culturing” refers to the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells may be cultured in suspension or while attached to a solid substrate. As used herein, “cell culture” and “cell culture broth” refer to the fluid contents of the bioreactor including the cells, cell culture media, metabolites and waste products.

[0161] As used herein, the terms “cell culturing medium“ (also called “culture medium”, “cell culture media”) refers to any nutrient solution used for growing cells, e.g., animal or mammalian cells, and which generally provides at least one or more components from the following: an energy source (usually in the form of a carbohydrate such as glucose); one or more of all essential amino acids, and generally the twenty basic amino acids, plus cysteine; vitamins and / or other organic compounds typically required at low concentrations; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.

[0162] Animal or mammalian cells are cultured in a medium suitable for the particular cells being cultured and which can be determined by the person of skill in the art without undue experimentation. "Serum-free" applies to a cell culture medium that does not contain animal sera, such as fetal bovine serum.

[0163] Commercially available media can be utilized and include, but is not limited to, Iscove’s Modified Dulbecco’s Medium, RPMI 1640, Minimal Essential Medium-alpha. (MEM-alpha), Dulbecco’s Modification of Eagle’s Medium (DMEM), DME / F12, alpha MEM, Basal Medium Eagle with Earle’s BSS, DMEM high Glucose, with Glutamine, DMEM high glucose, without Glutamine,DMEM low Glucose, without Glutamine, DMEM:F12 1: 1, with Glutamine, GMEM (Glasgow’s MEM), GMEM with glutamine, Grace’s Complete Insect Medium, Grace’s Insect Medium, without FBS, Flam’s F-10, with Glutamine, Flam’s F-12, with Glutamine, IMDM with HEPES and Glutamine, IMDM with HEPES and without Glutamine, IP41 Insect Medium, 15 (Leibovitz) (2x), without Glutamine or Phenol Red, 15 (Leibovitz), without Glutamine, McCoy’s 5A Modified Medium, Medium 199, MEM Eagle, without Glutamine or Phenol Red (2x), MEM Eagle-Earle’s BSS, with glutamine, MEM Eagle-Earle’s BSS, without Glutamine, MEM Eagle-Flanks BSS, without Glutamine, NCTC-I09, with Glutamine, Richter’s CM Medium, with Glutamine, RPMI 1640 with HEPES, Glutamine and / or Penicillin-Streptomycin, RPMI 1640, with Glutamine, RPMI 1640, without Glutamine, Schneider’s Insect Medium or any other media known to one skilled in the art, which are formulated for particular cell types. To the foregoing exemplary media can be added supplementary components or ingredients, including optional components, in appropriate concentrations or amounts, as necessary or desired, and as would be known and practiced by those having in the art using routine skill. Proprietary cell culture media can also be used.

[0164] The nutrient solution may optionally be supplemented with additional components to optimize growth of cells, such as hormones and other growth factors, such as insulin, transferrin, epidermal growth factor, serum, and the like; salts, such as calcium, magnesium and phosphate, and buffers, e.g., HEPES; nucleosides and bases, such as adenosine, thymidine, hypoxanthine; and protein and tissue hydrolysates, such as hydrolyzed plant or animal protein (peptone or peptone mixtures, which can be obtained from animal byproducts, purified gelatin or plant material); antibiotics, such as gentamycin; polyamines, such as putrescine, spermidine and spermine (see WIPO Publication No. WO 2008 / 154014) and pyruvate (see U.S. Pat. No. 8,053,238), antiapoptotic compounds, e.g., MDL 28170, cypermethrin, cyclosporine A, BBMP, Bongkrekic acid, S-15176 difumarate, cyclic pifithrin- a, pifithrin mu, BI-6C9, NSCI, NS3694 or Necrostatin-1 (see WIPO Publication No. WO 2014 / 022102) depending on the requirements of the cells to be cultured and / or the desired cell culture parameters.

[0165] A “perfusion” cell culture medium refers to a cell culture medium that is typically used in cell cultures that are maintained by perfusion or continuous culture methods and is sufficiently complete to support the cell culture during this process. Perfusion cell culture medium formulations may be richer or more concentrated than base cell culture medium formulations to accommodate themethod used to remove the spent medium. Perfusion cell culture medium can be used during both the growth and production phases.

[0166] A “production” cell culture medium refers to a cell culture medium that is typically used in cell cultures during the transition when exponential growth is ending and protein production takes over, “transition” and / or “product” phases, and is sufficiently complete to maintain a desired cell density, viability and / or product titer during this phase.

[0167] In any of the methods described herein, the culturing step can be perfusion culturing. As is known in the art, perfusion culturing includes removing from a bioreactor a first volume of a first liquid medium, and adding to the bioreactor a second volume of a second liquid medium, wherein the first volume and the second volume are about equal. The mammalian cells are retained in the bioreactor by some cell retention device or through techniques, such as cell settling in a settling cone.

[0168] As is known in the art, perfusion culturing is different than feed batch culturing, and the culture media and / or the culturing conditions used in perfusion culturing are often different than those used in feed batch culturing.

[0169] The removal and addition of media in perfusion culturing can be performed simultaneously or sequentially, or some combination of the two. Further, removal and addition can be performed continuously, such as at a rate that removes and replaces a volume of between 0. 1% to 800%, between 1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and 400%, between 1% and 350%, between 1% and 300%, between 1% and 250%, between 1% and 100%, between 100% and 200%, between 5% and 150%, between 10% and 50%, between 15% and 40%, between 8% and 80%, or between 4% and 30% of the volume of the bioreactor.

[0170] The first volume of the first liquid medium removed and the second volume of the second liquid medium added can in some instances be held approximately the same over each 24-hour period. As is known in the art, the rate at which the first volume of the first liquid medium is removed (volume / unit of time) and the rate at which the second volume of the second liquid medium is added (volume / unit of time) can be varied and depends on the conditions of the particular cell culture system. The rate at which the first volume of the first liquid medium is removed (volume / unit of time) and the rate at which the second volume of the second liquid medium is added (volume / unit of time) can be about the same or can be different.

[0171] Alternatively, the volume removed and added can change by gradually increasing over each 24-hour period. For example, the volume of the first liquid medium removed and the volume of the second liquid medium added within each 24-hour period can be increased over the culturing period. The volume can be increased over the culturing period from a volume that is between 50% to about 80% of the bioreactor volume. The volume can be increased over the culturing period to about 90% to about 100% of the bioreactor capacity or the volume of the cell culture at the start of the culturing period.

[0172] In some examples of the methods described herein, after the first 48 to 96 hours of the culturing period, in each 24-hour period (within the culturing period), the first volume of the first liquid medium removed and the second volume of the second liquid medium added is about 10% to about 95%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 85% to about 95%, about 60% to about 80%, or about 70% of the volume of the cell culture at the start of the culturing period.

[0173] Skilled practitioners will appreciate that the first liquid medium and the second liquid medium can be the same type of media. In other instances, the first liquid medium and the second liquid medium can be different. The second liquid medium may be more concentrated with respect to one or more media components.

[0174] Concentrated cell culture medium can contain some or all of the nutrients necessary to maintain the cell culture; in particular, concentrated medium can contain nutrients identified as or known to be consumed during the course of the production phase of the cell culture. Concentrated medium may be based on just about any cell culture media formulation. Such a concentrated feed medium can contain some or all the components of the cell culture medium at, for example, about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, lOOx, 200X, 400X, 600X, 800X, or even about 1000X of their normal amount.

[0175] The components used to prepare cell culture medium may be completely milled into a powder medium formulation; partially milled with liquid supplements added to the cell culture medium as needed; or added in a completely liquid form to the cell culture.

[0176] Cell cultures can also be supplemented with independent concentrated feeds of particular nutrients which may be difficult to formulate or are quickly depleted in cell cultures. Such nutrients may be amino acids such as tyrosine, cysteine and / or cystine (see e.g., International Patent Application Publication No. 2012 / 145682). For example, a concentrated solution of tyrosine can independently be fed to a cell culture grown in a cell culture medium containing tyrosine, such that the concentration of tyrosine in the cell culture does not exceed 8 mM. In another example, a concentrated solution of tyrosine and cystine is independently fed to the cell culture being grown in a cell culture medium lacking tyrosine, cystine or cysteine. The independent feeds can begin prior to or at the start of the production phase. The independent feeds can be accomplished by fed batch to the cell culture medium on the same or different days as the concentrated feed medium. The independent feeds can also be perfused on the same or different days as the perfused medium.

[0177] As used herein, the term “host cell” is understood to include a cell that has been genetically engineered to express a polypeptide of interest. Genetically engineering a cell involves transfecting, transforming or transducing the cell with a nucleic acid encoding a recombinant polynucleotide molecule (a “gene of interest”), and / or otherwise altering (e.g., by homologous recombination and gene activation or fusion of a recombinant cell with a non-recombinant cell) so as to cause the host cell to express a desired recombinant polypeptide. Methods and vectors for genetically engineering cells and / or cell lines to express a polypeptide of interest are well known to those of skill in the art; for example, various techniques are illustrated in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, R.J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic makeup to the original parent cell, so long as the gene of interest is present. A cell culture can comprise one or more host cells.

[0178] A wide variety of mammalian cell lines suitable for growth in the systems and methods disclosed herein are available from the American Type Culture Collection (Manassas, Va.) and commercial vendors. Examples of cell lines commonly used in the industry include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al, J. Gen Virol. 36: 59, 1977); babyhamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23 : 243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y Acad. Sci. 383:44-68, 1982); MRC 5 cells or ES4 cells; mammalian myeloma cells, and a number of other cell lines and Chinese hamster ovary (CHO) cells.

[0179] Large-scale production of proteins for commercial applications is typically carried out in suspension culture. Therefore, mammalian host cells used to generate the recombinant mammalian cells described herein can, but need not be, adapted to growth in suspension culture. A variety of host cells adapted to growth in suspension culture are known, including mouse myeloma NS0 cells and CHO cells from CHO-S, DG44, and DXB11 cell lines. Other suitable cell lines include mouse myeloma SP2 / 0 cells, baby hamster kidney BL1K-21 cells, human PER.C6® cells, human embryonic kidney L1EK-293 cells, and cell lines derived or engineered from any of the cell lines disclosed herein.

[0180] CHO cells are widely used to produce complex recombinant proteins, including CHOK1 cells (ATCC CCL61). The dihydrofolate reductase (DHLR)-deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77: 4216-4220), DXB11 and DG-44, are desirable CHO host cell lines because the efficient DHLR selectable and amplifiable gene expression system allows high level recombinant protein expression in these cells (Kaufman R. J. (1990), Meth Enzymol 185:537-566). Also included are the glutamine synthase (GS)-knockout CHOK1SV cell lines, making use of glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection. Other suitable CHO host cells could include, but are not limited to the following (ECACC accession numbers in brackets): CHO (85050302), CHO (PROTEIN TREE) (00102307), CHO-K1 (85051005), CHO-K1 / SF (93061607), CHO / dhFr-(94060607), CHO / dhFr-AC-free (05011002), RR-CHOKI (92052129).

[0181] Critical attributes and performance parameters of the cell culture can be measured to better inform decisions regarding performance of each step during manufacture. These critical attributes and parameters can be monitored real-time, near real-time, and / or following a unit operation. Critical parameters that can be measured during cell culture include, but are not limited to,cell culture media components that are consumed (such as, e.g., glucose), levels of metabolic byproducts (such as, e.g., lactate and ammonia) that accumulate, as well as parameters related to cell maintenance and survival, such as, e.g., dissolved oxygen content. Critical attributes such as specific productivity, viable cell density, packed cell volume, pH, osmolality, appearance, color, aggregation, percent yield, and titer may also be monitored during appropriated stages in the manufacturing process. Monitoring and measurements can be performed using known techniques and commercially available equipment.DOWNSTREAM OPERATIONS

[0182] The harvesting operation fully or partially clarifies and / or purifies the target protein away from at least one impurity with which it is found in the cell culture fluid, such as remaining cell culture media, cells, cell debris, undesired cell, or media components, and / or product- and / or process- related impurities. The recombinant protein can be purified from the harvest permeate by one or more of flocculation, precipitation, centrifugation, depth filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode anion exchange chromatography, hydrophobic interaction chromatography or hydroxyapatite chromatography. In one sub-embodiment, the methods of the invention further comprise taking samples during the purification process, evaluating the samples to quantitatively and / or qualitatively monitor characteristics of the recombinant protein and the purification process. The samples can be quantitatively and / or qualitatively monitored using process analytical techniques.

[0183] The recombinant protein can be formulated into a pharmaceutically acceptable formulation. “Formulated” generally means buffer exchanged, sterilized, bulk-packaged, and / or packaged for a final user. Suitable formulations for pharmaceutical compositions include those described in Remington ’s Pharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton, Pa.PROTEINS OF INTEREST

[0184] The present disclosure provides methods for culturing cells expressing a “protein of interest” or a “recombinant protein” and such terms are generally used interchangeably. A protein of interest or recombinant protein can, but need not be, a protein that is known or suspected to be therapeutically relevant. Particular examples of a protein of interest or recombinant protein includeantigen binding proteins (as described and defined herein), peptibodies (z.e., a molecule comprising peptide(s) fused either directly or indirectly to other molecules such as an Fc domain of an antibody, where the peptide moiety specifically binds to a desired target; the peptide(s) may be fused to either an Fc region or inserted into an Fc-Loop, or a modified Fc molecule, for example as described in U.S. Patent Application Publication No. US2006 / 0140934 incorporated herein by reference in its entirety), fusion proteins (e.g., Fc fusion proteins, wherein a Fc fragment is fused to a protein or peptide, including a peptibody), cytokines, growth factors, hormones and other naturally occurring secreted proteins, as well as mutant forms of naturally occurring proteins.

[0185] Proteins of interest and recombinant proteins include “antigen-binding proteins”. Antigen-binding protein refers to proteins or polypeptides that comprise an antigen-binding region or antigen-binding portion that has affinity for another molecule to which it binds (antigen). An antigen binding protein can have one or more binding sites. If there is more than one binding site, the binding sites can be identical to one another or can be different. For example, a naturally occurring human immunoglobulin typically has two identical binding sites, while a “bispecific” or “bifimctional” antibody has two different binding sites.

[0186] Antigen-binding proteins encompass antibodies, peptibodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including single-chain variable fragments (scFvs), double-chain (divalent) scFvs, and IgGscFv (see, e.g., Orcutt et al., 2010, Protein Eng Des Sei 23:221-228), hetero-IgG (see, e.g., Liu et al., 2015, J Biol Chem 290:7535-7562), muteins, and XmAb® (Xencor, Inc., Monrovia, CA). Also included are bispecific T cell engagers (BiTE®), bispecific T cell engagers having extensions, such as half life extensions, for example HLE BiTEs, Heterolg BITE and others, chimeric antigen receptors (CARs, CAR Ts), and T cell receptors (TCRs).

[0187] As used herein, the term “antigen binding protein” is used in its broadest sense and means a protein comprising a portion that binds to an antigen or target and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include a human antibody, a humanized antibody; a chimeric antibody; a recombinant antibody; a single chain antibody; a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab’)2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgGl antibody; an IgG2 antibody; an IgG3 antibody; or an IgG4 antibody, and fragments thereof. The antigen binding protein can comprise, for example, analternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, e.g., Komdorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). In addition, peptide antibody mimetics (“PAMs”) can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components as a scaffold.

[0188] The term “antibody” includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. Unless otherwise specified, antibodies include human, humanized, chimeric, multi-specific, monoclonal, polyclonal, heteroIgG, bispecific. Antibodies include the IgGl-, lgG2- lgG3- or lgG4-type.

[0189] An “immunoglobulin” is a tetrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively.

[0190] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain can be done in accordance with the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, 5thEd., US Dept, of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, (1991). As desired, the CDRs can also be redefined according to an alternative nomenclature scheme, such as that of Chothia (see Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883 or Honegger & Pluckthun, 2001, J. Mol. Biol. 309:657-670).

[0191] Also included are modified proteins, such as are proteins modified chemically by a non- covalent bond, covalent bond, or both a covalent and non-covalent bond. Also included are proteins further comprising one or more post-translational modifications which may be made by cellular modification systems or modifications introduced ex vivo by enzymatic and / or chemical methods or introduced in other ways.

[0192] Proteins of interest and recombinant proteins may also include recombinant fusion proteins comprising, for example, a multimerization domain, such as a leucine zipper, a coiled coil, an Fc portion of an immunoglobulin, and the like. Also included are proteins comprising all or part of the amino acid sequences of differentiation antigens (referred to as CD proteins) or their ligands or proteins substantially similar to either of these.

[0193] For purposes of clarity, and as described herein, it is noted that an antigen binding protein can, but need not, be of human origin (e.g., a human antibody), and in some cases will comprise non-human sequences, for example a rat or murine sequence, and in other cases an antigen binding protein can comprise a hybrid of human and non-human sequences (e.g., a humanized antibody).

[0194] A protein of interest and recombinant protein can comprise a human antibody. The term “human antibody” includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (a fully human antibody). Such antibodies can be prepared in a variety of ways, including through the immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and / or light chain-encoding genes, such as a mouse derived from a Xenomouse®, UltiMab™, or Velocimmune® system. Phage-based approaches can also be employed.

[0195] Alternatively, a protein of interest and recombinant protein can comprise a humanized antibody. A “humanized antibody” has a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions, such that the humanized antibody is less likely to induce an immune response, and / or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject. Certain amino acids in the framework and constant domains of the heavy and / or light chains of the non-human species antibody can be mutated to produce thehumanized antibody. Alternatively, the constant domain(s) from a human antibody can be fused to the variable domain(s) of a non-human species. Examples of how to make humanized antibodies can be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.

[0196] Antigen binding portions include, inter alia, Fab, Fab’, F(ab’)2, Fd, Fv, domain antibodies (dAbs), fragments including complementarity determining regions (CDRs), single-chain antibodies (scFv), single domain VHH, chimeric antibodies, diabodies, maxibodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Antigen binding portions or fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies and can form an element of a protein of interest.

[0197] A Fab fragment is a monovalent fragment having the VL, VH, CL and Cnl domains; a F(ab’)2 fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the VH and CRI domains; an Fv fragment has the VL and VH domains of a single arm of an antibody; and a dAb fragment has a VH domain, a VL domain, or an antigenbinding fragment of a VH or VL domain (U.S. Pat. Nos. 6,846,634, 6,696,245, U.S. App. Pub. Nos. 2005 / 0202512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, 2003 / 0039958, Ward et al., 1989, Nature 341:544-546).

[0198] A single-chain antibody (scFv) is an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48; and Poljak et al., 1994, Structure 2: 1121-23). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies areantibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.

[0199] One or more CDRs of an antibody can be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein. An antigen binding protein can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the CDR(s) noncovalently. The CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest.

[0200] An “Fc” region, as the term is used herein, comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. Proteins of interest comprising an Fc region, including antigen binding proteins and Fc fusion proteins, form another aspect of the instant disclosure.

[0201] A “hemibody” is an immunologically functional immunoglobulin construct comprising a complete heavy chain, a complete light chain and a second heavy chain Fc region paired with the Fc region of the complete heavy chain. A linker can, but need not, be employed to join the heavy chain Fc region and the second heavy chain Fc region. In particular sub-embodiments, a hemibody is a monovalent form of an antigen binding protein disclosed herein. In other sub-embodiments, pairs of charged residues can be employed to associate one Fc region with the second Fc region.

[0202] Polypeptides and proteins of interest and recombinant proteins can be of scientific or commercial interest, including protein-based therapeutics. Proteins of interest and recombinant proteins include, among other things, secreted proteins, non-secreted proteins, intracellular proteins or membrane-bound proteins. Polypeptides and proteins of interest can be produced by recombinant animal cell lines using cell culture methods and may be referred to as “recombinant proteins”. The expressed protein(s) may be produced intracellularly or secreted into the culture medium from which it can be recovered and / or collected. The term “isolated protein” or “isolated recombinant protein” refers to a polypeptide or protein of interest, that is purified away from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research or other use. Proteins of interest and recombinant proteins include proteins that exert a therapeutic effect by binding a target, particularly a target among those listed below, including targets derived therefrom, targets related thereto, and modifications thereof.

[0203] In some embodiments, proteins of interest and recombinant proteins may include colony stimulating factors, such as granulocyte colony-stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to, Neupogen® (fdgrastim) and Neulasta® (pegfilgrastim). Also included are erythropoiesis stimulating agents (ESA), such as Epogen® (epoetin alfa), Aranesp® (darbepoetin alfa), Dynepo® (epoetin delta), Mircera® (methyoxy polyethylene glycol-epoetin beta), Hematide®, MRK-2578, INS-22, Retacrit® (epoetin zeta), Neorecormon® (epoetin beta), Silapo® (epoetin zeta), Binocrit® (epoetin alfa), epoetin alfa Hexal, Abseamed® (epoetin alfa), Ratioepo® (epoetin theta), Eporatio® (epoetin theta), Biopoin® (epoetin theta), epoetin alfa, epoetin beta, epoetin zeta, epoetin theta, and epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activator, GLP-1 receptor agonists, as well as the molecules or variants or analogs thereof and biosimilars of any of the foregoing.

[0204] In some sub-embodiments, proteins of interest and recombinant proteins bind to one of more of the following, alone or in any combination: CD proteins, including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174; HER receptor family proteins, including, for instance, HER2, HER3, HER4, and the EGF receptor, EGFRvIII; cell adhesion molecules, for example, LFA-1, Mol, pl 50,95, VLA-4, ICAM-1, VCAM, and alpha v / beta 3 integrin; growth factors, including but not limited to, for example, vascular endothelial growth factor (“VEGF”); VEGFR2, growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, mullerian-inhibiting substance, human macrophage inflammatory protein (MIP-1 -alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), Cripto, transforming growth factors (TGF), including, among others, TGF-a and TGF-J3, including TGF-J31, TGF-J32, TGF-J33, TGF-J34, or TGF-J35, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-I), and osteoinductive factors; insulins and insulin-related proteins, including but not limited to insulin, insulin A-chain, insulin B- chain, proinsulin, and insulin-like growth factor binding proteins; coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrand factor, protein C, alpha- 1- antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator (“t-PA”), bombazine, thrombin, thrombopoietin, and thrombopoietin receptor; colony stimulating factors(CSFs), including the following, among others, M-CSF, GM-CSF, and G-CSF; other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens, receptors and receptor- associated proteins, including, for example, flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptors, and T-cell receptors; neurotrophic factors, including but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); relaxin A-chain, relaxin B-chain, and prorelaxin; interferons, including for example, interferon-alpha, -beta, and -gamma, interleukins (ILs), e.g., IL-1 to IL- 10, IL- 12, IL- 15, IL- 17, IL-23, IL-12 / IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2, or IL-17 receptor, IL-1RAP; viral antigens, including but not limited to, an AIDS envelope viral antigen; as well as any of the following: lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide, DNase, FR-alpha, inhibin, and activin, integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressins, regulatory proteins, immunoadhesins, somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c- Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGp, hepatitis-C virus, mesothelin dsFv[PE38] conjugate, Legionella pneumophila (lly), IFN gamma, interferon gamma induced protein 10 (IP 10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / Kexin Type 9 (PCSK9), stem cell factors, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, a4p7, platelet specific (platelet glycoprotein Ilb / IIIb (PAC-1), transforming growth factor beta (TFGP), Zona pellucida spermbinding protein 3 (ZP-3), TWEAK, platelet derived growth factor receptor alpha (PDGFRa), and sclerostin.

[0205] In another sub-embodiment, proteins of interest and recombinant proteins include abciximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosozumab, blinatumomab, brentuximab vedotin,brodalumab, cantuzumab mertansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lerdelimumab, lumiliximab, Ixdkizumab, mapatumumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelvekin, palivizumab, panitumumab, pembrolizumab, pertuzumab, pexelizumab, ranibizumab, rilotumumab, rituximab, romiplostim, romosozumab, sargamostim, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, visilizumab, volociximab, zanolimumab, zalutumumab, and biosimilars of any of the foregoing.

[0206] Proteins of interest and recombinant proteins can also include genetically engineered receptors such as chimeric antigen receptors (CARs or CAR-Ts) and T cell receptors (TCRs), as well as other proteins comprising an antigen binding molecule that employ a similar targeting mechanism. CARs can be engineered to bind to an antigen (such as a cell-surface antigen) by incorporating an antigen binding molecule that interacts with that targeted antigen. CARs typically incorporate an antigen binding domain (such as scFv) in tandem with one or more costimulatory (“signaling”) domains and one or more activating domains.NON-LIMITING EXAMPLE EMBODIMENTS

[0207] Example embodiments / features of the present disclosure include, but are not limited to, the following Features 1-49.

[0208] Feature 1 : A method for producing a recombinant protein comprising: a) culturing mammalian cells expressing the recombinant protein in a bioreactor under perfusion and recirculating tangential flow (RTF) filtration conditions sufficient to produce the recombinant protein and at a working volume from 50% to 95% of the final working volume; b) maintaining the foam coverage at the fluid surface of the bioreactor at a target of 10% or less through the presence of antifoam; c) increasing the working volume to the final working volume wherein the cell culture within the bioreactor contains antifoam in an amount of at least 14 mg antifoam / kg of cell culture (mg / kg) at the initiation of increasing the workingvolume, wherein the amount of antifoam is calculated based on the final working volume; and d) culturing the mammalian cells for the duration of a production run.

[0209] Feature 2: The method of Feature 1, wherein the amount of antifoam at step c) is at least 24 mg / kg.

[0210] Feature 3: The method of Feature 1 or 2, wherein the culturing in step a) is with a bioreactor inoculated with 1 x 106to 20 x 106cells / ml.

[0211] Feature 4: The method of Feature 3, wherein the culturing in step a) is with a bioreactor inoculated 6 x 106to 14 x 106cells / ml.

[0212] Feature 5: The method of any of Features 1 to 4, wherein the increasing the working volume step is initiated between 24 to 120 hours after initiation of the culture.

[0213] Feature 6: The method of any of Features 1 to 5, wherein the increasing the working volume step is maintained for 24 to 48 hours.

[0214] Feature 7: The method of any of Features 1 to 6, wherein the culturing in step a) is at an working volume from 60% to 70% of the final working volume.

[0215] Feature 8: The method of any of Features 1 to 7, wherein the antifoam is added in a continuous or semi-continuous manner, or as a single bolus.

[0216] Feature 9: The method of Feature 8, wherein the antifoam is added to the bioreactor separately from the perfusion culture media.

[0217] Feature 10: The method of any of Features 1 to 9, wherein the recirculating tangential flow filtration is through a hollow fiber filter, a flat sheet membrane or a spiral -wound membrane.

[0218] Feature 11: The method of Feature 10, wherein the recirculating tangential flow filtration is through a hollow fiber filter.

[0219] Feature 12: The method of Feature 11, wherein the hollow fiber filter has a pore size or molecular weight cut off that retains the recombinant protein and cells in the bioreactor.

[0220] Feature 13: The method of Feature 11 or 12, wherein the hollow fiber filter that has a pore size or molecular weight cut off that retains the recombinant protein and cells in the bioreactor is an ultrafilter.

[0221] Feature 14: The method of Feature 13, wherein the molecular weight cutoff of the ultrafilter is 50 kDa.

[0222] Feature 15: The method of Feature 13 or 14, wherein the pore size of the ultrafilter is 0.01 micrometers to 0.1 micrometers.

[0223] Feature 16: The method of any of Features 1 to 15, wherein antifoam is added after the increasing the working volume step in an amount sufficient to maintain less than 50% foam coverage at the fluid surface of the bioreactor.

[0224] Feature 17: The method of Feature 16, wherein antifoam is added after the increasing the working volume step in an amount sufficient to maintain less than 10% foam coverage at the fluid surface of the bioreactor.

[0225] Feature 18: The method of Feature 16 or 17, wherein the amount of antifoam at the end of the culturing step d) is between 70 mg / kg and 300 mg / kg.

[0226] Feature 19: The method of any of Features 1 to 18, wherein the antifoam is simethicone.

[0227] Feature 20: The method of any of Features 1 to 19, wherein the bioreactor capacity ranges from 200L to 20000L.

[0228] Feature 21 : The method of any of Features 1 to 20, wherein the recombinant protein is an immunoglobulin, antibody, or antibody fragment.

[0229] Feature 22: The method of any of Features 1 to 21, wherein the duration of the production run is 10 to 40 days.

[0230] Feature 23: The method of any of Features 1 to 22, further comprising e) harvesting the recombinant protein.

[0231] Feature 24: The method of Feature 23, wherein harvesting includes filtering the permeate through a microfiltration (MF) filter.

[0232] Feature 25: The method of Feature 24, wherein the MF filter has a MWCO of 750 kDa.

[0233] Feature 26: The method of Feature 24 or 25, wherein the MF filter has a pore size of 0.2 pm.

[0234] Feature 27: The method of Feature 1, wherein the working volume in step a) is from 50% to 75% of the final working volume, and the amount of antifoam at step c) is in the range of 23 mg / kg to 38 mg / kg.

[0235] Feature 28: The method of any one of Features 1-27, wherein the antifoam is simethicone, and the amount of antifoam in the bioreactor when the culture is terminated or harvested is between 110 mg / kg and 180 mg / kg.

[0236] Feature 29: The method of any one of Features 1-3, wherein the culturing in step a) is with a bioreactor inoculated with 1 x 106to 20 x 106cells / ml, and the increasing the working volume step is initiated between 24 to 120 hours after initiation of the culturing in step a).

[0237] Feature 30: The method of any one of Features 1-29, wherein the antifoam is added in a continuous manner, and the amount of antifoam at step c) is in the range of 24 mg / kg to 38 mg / kg.

[0238] Feature 31: The method of any one of Features 1-29, wherein the antifoam is added as a bolus, and the amount of antifoam when the culture is terminated or harvested is between 90 mg / kg and 220 mg / kg.

[0239] Feature 32: The method of Feature 1, wherein the recombinant protein is an antibody; the working volume in step a) is from 50% to 75% of the final working volume; the amount of antifoam at step c) is in the range of 23 mg / kg to 38 mg / kg; the antifoam is simethicone; and the amount of antifoam in the bioreactor when the culture is terminated or harvested is between 90 mg / kg and 220 mg / kg.

[0240] Feature 33: The method of Feature 1, wherein the recombinant protein is an antibody; the working volume in step a) is from 50% to 75% of the final working volume; the amount of antifoam at step c) is in the range of 23 mg / kg to 38 mg / kg; the antifoam is simethicone; the culturing in step a) is with a bioreactor inoculated with 1 x 106to 20 x 106cells / ml; the increasing the working volume step is initiated between 24 to 120 hours after initiation of the culturing in step a); and the amount of antifoam in the bioreactor when the culture is terminated or harvested is between 90 mg / kg and 220 mg / kg.

[0241] Feature 34: A method for producing a recombinant protein comprising:initiating a culture in a bioreactor at a working volume that is at least 50% of a final working volume; inoculating the culture with mammalian cells engineered to express the recombinant protein; perfusing the culture at one or more perfusion rates of less than or equal to 0.5 culture volumes / day (V / d) until the culture reaches one or more desired target criteria, wherein foam coverage at the fluid surface of the bioreactor is maintained at 10% or less during the perfusing; increasing the working volume to the final working volume, wherein the bioreactor contains antifoam in an amount of at least 14 mg antifoam / kg of cell culture (mg / kg) at the initiation of increasing the working volume, wherein the amount of antifoam is calculated based on the final working volume; and once the final working volume is achieved, perfusing the culture at one or more perfusion rates of less than or equal to 0.5 culture volumes / day (V / d) until the culture is terminated or harvested, wherein all rates expressed in V / d are calculated based on the final working volume.

[0242] Feature 35: The method of Feature 34, wherein the working volume at the initiation of the culture is in the range of 50% to 75% of the final working volume.

[0243] Feature 36: The method of Feature 34 or Feature 35, wherein the mammalian cells are CHO cells.

[0244] Feature 37: The method of any one of Features 34-36, wherein the recombinant protein is an antibody.

[0245] Feature 38: The method of any one of Features 34-37, wherein the antifoam is simethicone.

[0246] Feature 39: The method of any one of Features 34-38, wherein the amount of antifoam at the initiation of increasing the culture volume is in the range of 23 mg / kg to 38 mg / kg.

[0247] Feature 40: The method of any one of Features 34-39, wherein the working volume at the initiation of the culture is 50% to 75% of the final working volume, the mammalian cells are CHOcells, the recombinant protein is an antibody, the antifoam is simethicone, and the amount of antifoam at the initiation of increasing the working volume is in the range of 23 mg / kg to 38 mg / kg.

[0248] Feature 41 : A method for producing a recombinant protein comprising: initiating a culture in a bioreactor at a working volume that is at least 50% of a final working volume; inoculating the culture with mammalian cells engineered to express the recombinant protein; perfusing the culture for 24 hours to 72 hours at one or more perfusion rates of 0.05 V / d to 0.5 V / d, wherein foam coverage at the fluid surface of the bioreactor is maintained at 10% or less during the perfusing; increasing the working volume to the final working volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates, wherein at least one of the one or more feed rates is less than or equal to 0.50 V / d and at least one of the one or more permeate rates is less than or equal to 0.20 V / d, wherein each of the one or more feed rates is greater than the contemporaneous permeate rate and further wherein the bioreactor contains simethicone in an amount of at least 14 mg simethicone / kg of cell culture (mg / kg) at the initiation of increasing the working volume, wherein the amount of simethicone is calculated based on the final working volume; and once the final working volume is achieved, perfusing the culture at one or more perfusion rates of less than or equal to 0.5 culture volumes / day (V / d) until the culture is terminated or harvested, wherein all rates expressed in V / d are calculated based on the final working volume.

[0249] Feature 42: The method of Feature 41, wherein the working volume at the initiation of the culture is 50% to 75% of the final working volume.

[0250] Feature 43: The method of Feature 41 or Feature 42, wherein the mammalian cells areCHO cells.

[0251] Feature 44: The method of any one of Features 41-43, wherein the recombinant protein is an antibody.

[0252] Feature 45: The method of any one of Features 41-44, wherein the one or more perfusion rates during the 24 to 72 hour perfusing step are in the range of 0. 10 V / d to 0.25 V / d.

[0253] Feature 46: The method of any one of Features 41-45, wherein the amount of simethicone at the initiation of increasing the culture volume is in the range of 23 mg / kg to 38 mg / kg.

[0254] Feature 47: The method of any one of Features 41-46, wherein the amount of simethicone in the bioreactor when the culture is terminated or harvested is between 80 mg / kg and 240 mg / kg.

[0255] Feature 48: The method of any one of Features 41-47, wherein the amount of simethicone in the bioreactor when the culture is terminated or harvested is between 100 mg / kg and 150 mg / kg.

[0256] Feature 49: The method of any one of Features 41-48, wherein the working volume at the initiation of the culture is 50% to 75% of the final working volume, the mammalian cells are CHO cells, the recombinant protein is an antibody, the one or more perfusion rates during the 24 to 72 hour perfusing step are in the range of 0.10 V / d to 0.25 V / d, the amount of simethicone at the initiation of increasing the working volume is in the range of 23 mg / kg to 38 mg / kg, and the amount of simethicone in the bioreactor when the culture is terminated or harvested is between 100 mg / kg and 150 mg / kg.

[0257] The present invention is not to be limited in scope by the specific embodiments described herein that are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.EXAMPLESEXAMPLE 1: 500 L EXPERIMENTS TO COMPARE RTF AND ATF FILTRATION SYSTEMS

[0258] For these experiments, the RTF assembly utilized an entirely single-use autoclaved custom-made assembly similar in design to that shown in FIG. 1A. The assembly was constructed with a primary and backup filter. An ATF assembly employed for these experiments utilized an assembly similar in design to that shown in FIG. IB. The filters used for this experiment were Spectrum KrosFlo 50 kDa (RTF) membranes and a Cytiva RTP ATF4 (ATF) filter (0.84 sqm. surface area).

[0259] For each run, the production bioreactor process begins with inoculation with mAb- producing CHO cells at a working volume that is approximately 68% of the final working volume in a proprietary cell culture medium. The perfusion process is initiated approximately one day after inoculation at a exchange rate of 0.13 culture volumes per day (CV / day, also referred to herein as V / day or V / d), where culture volume refers to the bioreactor’s final working volume. Three days into the culture, the working volume is gradually increased over a 30-hour period to the final working volume while the permeate rate maintained at 0.13 CV / day. Once this has been completed, level control at the final working volume is enabled. The perfusion rate is then increased to 0.22 CV / day from day 5 through to the end of the process. 1% simethicone was present in the cell culture media at a concentration less than or equal to 0.75 mL / L (approximately 5.25 to 11.25 mg / kg).

[0260] Visual inspection of the filter lumens indicated that the inlets to the RTF filters were heavily fouled by cell debris shortly after feed-up initiation. The first filter fouled immediately after the conclusion of the feed-up portion of the process and the second filter fouled shortly thereafter. FIGs. 2A and 2B show the feed pressure from the duration of both 500 L experiments as a pre- antifoam schedule implementation. The increased pressure indicates the filter fouling as the cell culture material is unable to enter the filter. Debris was determined through Fourier Transform Infrared (FTIR) Spectroscopy to be highly proteinaceous in makeup.

[0261] An observation was made that cell culture debris tended to accumulate on the bioreactor walls at the surface of the culture prior to feed-up. While crust layer formation is not atypical or problematic for other perfusion processes in which the working volume is held constant, this was not the case for the feed-up strategy that changes the working volume. It was then hypothesized that, when the feed-up begins, desiccated cell debris is washed back into the culture. This hard-to-dissolve material is then pulled into the RTF recirculation loop where it eventually fouls the RTF HFM.EXAMPLE 2: ROOT CAUSE IDENTIFICATION

[0262] A series of small-scale experiments and pilot scale runs were executed to determine contributing factors to the observed performance issues and fouling, as well as to investigate process mitigations to improve RTF robustness.

[0263] The bioreactor process conditions were identical to Example 1 except at a smaller scale.

[0264] A root cause analysis concluded that the most probable causes for the challenges were related to lumen specific crossflow rate and the control of foam on the surface of the culture prior to the feed-up. Controlling foam buildup on the reactor is instrumental to RTF success as it has been found to prevent the accumulation of debris on the sidewalls of the bioreactor vessel. When foam is allowed to accumulate, cell debris is allowed to remain at the surface of the culture for a longer period, where it desiccates and forms a “crust layer” that is difficult to dissolve. When the feed-up begins, this crust layer is introduced into the bioreactor’s working volume and eventually accumulates at the inlet of the RTF filter, leading to catastrophic filter fouling. If a single RTF HFM is fouled through the mechanism described, it is likely that an additional filter, if added, would also foul as the debris cannot be cleared from the culture.

[0265] Further experiments were conducted at small-scale which used antifoam to prevent any foam from building up on the surface of the culture. Results from these small-scale experiments indicated that the crust layer could be minimized through addition of antifoam.EXAMPLE 3: INTRODUCTION OF ANTIFOAM: PILOT SCALE

[0266] Pilot-scale runs were performed using antifoam to prevent any foam from building up on the surface of the culture. The goal was to ensure that less than 10% of the cell culture surface was covered in foam. Instructions were provided to dose antifoam as needed to ensure that no foam was visible on the culture surface, in order to test the hypothesis that minimizing foam accumulation could help mitigate fouling at the fdter inlet.

[0267] The bioreactor process conditions were identical to Example 1. Prior to the beginning of the feed-up, higher amounts of 1% simethicone were added to ensure minimal foam is visible at the surface. The 1% solution was added to the cell culture media in the bioreactor at various amounts as shown in FIGs. 3A and 3B.

[0268] Feed pressure, permeate pressure, and transmembrane pressure (TMP) were measured during the process. FIGs. 2A and 2B show how system inlet pressure compared to prior experiments executed at 500 L. Feed pressure and differential pressure (Feed Flow Pressure - Retentate Pressure) increased gradually throughout the duration of the process, indicating that minimal fouling had occurred.

[0269] After the successful completion of the run, which exhibited well-controlled pressure and relatively low levels of debris accumulation, totalized antifoam throughout the duration of the process was used to build out an automated antifoam addition schedule. A similar antifoam schedule was utilized and refined over a series of later RTF pilot-scale runs.

[0270] Following the conclusion of the bioreactor process, three additional antibody processes were also executed using a similar antifoam schedule. System pressures remained stable throughout these processes as well. Accordingly, this RTF antifoam strategy has been demonstrated for four different molecules across a range of bioreactor scales and formats.

[0271] FIGs. 3A and 3B show a compilation of a number of bioreactor process runs showing successful and unsuccessful reduction of filter fouling. Based on pilot scale data for several runs, 1% solution added to achieve an amount of antifoam between 2.39 mL / kg and 3.72 mL / kg (23.9 ppm - 37.2 ppm) was able to reduce filter fouling prior to initiation of feed up.EXAMPLE 4: ANTIFOAM IN ADDITIONAL SETTINGS

[0272] FIGs. 4A and 4B compare feed flow (FIG. 4A) and permeate (FIG. 4B) pressures in RTF runs across multiple scales that have used various iterations of the RTF antifoam strategy in order to meet the low foam criteria. Use of the proposed antifoam strategy has enabled robust control of pressures relative to pre-defined limits and successful completion of runs without requiring RTF filter change out, for N-0 scales ranging from 200 L to 2000 L in both stainless steel and single-use bioreactor formats.EXAMPLE 5: CELL CULTURE PERFORMANCE

[0273] Experiments executed at the pilot scale have shown that cell culture performance and product quality are highly comparable for RTF versus ATF. Illustratively, viable cell density (VCD),culture viability, and packed cell volume (PCV) adjusted titer from a number of mAb runs with ATF and RTF fdtration perfusion processes are shown in FIGs. 5A-5C.

[0274] Initial data indicate that viabilities can be lower for perfusion processes using RTF versus ATF fdtration, potentially caused by different rates of cell shear associated with the RTF feed pump; however, data from various scales show that the overall productivity remains comparable.IQ

Claims

What is claimed is:

1. A method for producing a recombinant protein comprising: a. culturing mammalian cells expressing the recombinant protein in a bioreactor under perfusion and recirculating tangential flow (RTF) fdtration conditions sufficient to produce the recombinant protein and at a working volume from 50% to 95% of the final working volume; b. maintaining the foam coverage at the fluid surface of the bioreactor at a target of 10% or less through the presence of antifoam; c. increasing the working volume to the final working volume wherein the cell culture within the bioreactor contains antifoam in an amount of at least 14 mg antifoam / kg of cell culture (mg / kg) at the initiation of increasing the volume, wherein the amount of antifoam is calculated based on the final working volume; and d. culturing the mammalian cells for the duration of a production run.

2. The method of claim 1, wherein the amount of antifoam at step c) is at least 24 mg / kg.

3. The method of claim 1 or 2, wherein the culturing in step a) is with a bioreactor inoculated with 1 x 106to 20 x 106cells / ml.

4. The method of claim 3, wherein the culturing in step a) is with a bioreactor inoculated 6 x 106to14 x 106cells / ml.

5. The method of any one of claims 1 to 4, wherein the increasing the working volume step is initiated between 24 to 120 hours after initiation of the culture.

6. The method of any one of claims 1 to 5, wherein the increasing the working volume step is maintained for 24 to 48 hours.

7. The method of any one of claims 1 to 6, wherein the culturing in step a) is at an working volume from 60% to 70% of the final working volume.

8. The method of any one of claims 1 to 7, wherein the antifoam is added in a continuous or semi- continuous manner, or as a single bolus.

9. The method of claim 8, wherein the antifoam is added to the bioreactor separately from the perfusion culture media.

10. The method of any one of claims 1 to 9, wherein the recirculating tangential flow filtration is through a hollow fiber filter, a flat sheet membrane or a spiral-wound membrane.

11. The method of claim 10, wherein the recirculating tangential flow filtration is through a hollow fiber filter.

12. The method of claim 11, wherein the hollow fiber filter has a pore size or molecular weight cut off that retains the recombinant protein and cells in the bioreactor.

13. The method of claim 11 or 12, wherein the hollow fiber filter that has a pore size or molecular weight cut off that retains the recombinant protein and cells in the bioreactor is an ultrafilter.

14. The method of claim 13, wherein the molecular weight cutoff of the ultrafilter is 50 kDa.

15. The method of claim 13 or 14, wherein the pore size of the ultrafilter is 0.01 micrometers to 0. 1 micrometers.

16. The method of any one of claims 1 to 15, wherein antifoam is added after the increasing the working volume step in an amount sufficient to maintain less than 50% foam coverage at the fluid surface of the bioreactor.

17. The method of claim 16, wherein antifoam is added after the increasing the working volume step in an amount sufficient to maintain less than 10% foam coverage at the fluid surface of the bioreactor.

18. The method of claim 16 or 17, wherein the amount of antifoam at the end of the culturing step d) is between 70 mg / kg and 300 mg / kg.

19. The method of any one of claims 1 to 18, wherein the antifoam is simethicone.

20. The method of any one of claims 1 to 19, wherein the bioreactor capacity ranges from 200L to 20000L.

21. The method of any one of claims 1 to 20, wherein the recombinant protein is an immunoglobulin, antibody, or antibody fragment.

22. The method of any one of claims 1 to 21, wherein the duration of the production run is 10 to 40 days.

23. The method of any one of claims 1 to 22, further comprising e. harvesting the recombinant protein.

24. The method of claim 23, wherein harvesting includes filtering the permeate through a microfdtration (MF) fdter.

25. The method of claim 24, wherein the MF filter has a MWCO of 750 kDa.

26. The method of claim 24 or 25, wherein the MF filter has a pore size of 0.2 pm.

27. The method of claim 1, wherein the working volume in step a) is from 50% to 75% of the final working volume, and the amount of antifoam at step c) is in the range of 23 mg / kg to 38 mg / kg.

28. The method of any one of claims 1-27, wherein the antifoam is simethicone, and the amount of antifoam in the bioreactor when the culture is terminated or harvested is between 110 mg / kg and 180 mg / kg.

29. The method of any one of claims 1-3, wherein the culturing in step a) is with a bioreactor inoculated with 1 x 106to 20 x 106cells / ml, and the increasing the working volume step is initiated between 24 to 120 hours after initiation of the culturing in step a).

30. The method of any one of claims 1-29, wherein the antifoam is added in a continuous manner, and the amount of antifoam at step c) is in the range of 24 mg / kg to 38 mg / kg .

31. The method of any one of claims 1-29, wherein the antifoam is added as a bolus, and the amount of antifoam when the culture is terminated or harvested is between 90 mg / kg and 220 mg / kg.

32. The method of claim 1, wherein the recombinant protein is an antibody; the working volume in step a) is from 50% to 75% of the final working volume; the amount of antifoam at step c) is in the range of 23 mg / kg to 38 mg / kg; the antifoam is simethicone; and the amount of antifoam in the bioreactor when the culture is terminated or harvested is between 90 mg / kg and 220 mg / kg.

33. The method of claim 1, wherein the recombinant protein is an antibody; the working volume in step a) is from 50% to 75% of the final working volume; the amount of antifoam at step c) is in the range of 23 mg / kg to 38 mg / kg; the antifoam is simethicone; the culturing in step a) is with a bioreactor inoculated with 1 x 106to 20 x 106cells / ml; the increasing the working volume step is initiated between 24 to 120 hours after initiation of the culturing in step a); and the amount of antifoam in the bioreactor when the culture is terminated or harvested is between 90 mg / kg and 220 mg / kg.

34. A method for producing a recombinant protein comprising: initiating a culture in a bioreactor at a working volume that is at least 50% of a final working volume; inoculating the culture with mammalian cells engineered to express the recombinant protein; perfusing the culture at one or more perfusion rates of less than or equal to 0.5 culture volumes / day (V / d) until the culture reaches one or more desired target criteria, wherein foam coverage at the fluid surface of the bioreactor is maintained at 10% or less during the perfusing; increasing the working volume to the final working volume, wherein the bioreactor contains antifoam in an amount of at least 14 mg antifoam / kg of cell culture (mg / kg) at the initiation of increasing the working volume, wherein the amount of antifoam is calculated based on the final working volume; and once the final working volume is achieved, perfusing the culture at one or more perfusion rates of less than or equal to 0.5 V / d until the culture is terminated or harvested, wherein all rates expressed in V / d are calculated based on the final working volume.

35. The method of claim 34, wherein the working volume at the initiation of the culture is in the range of 50% to 75% of the final working volume.

36. The method of claim 34 or claim 35, wherein the mammalian cells are CHO cells.

37. The method of any one of claims 34-36, wherein the recombinant protein is an antibody.

38. The method of any one of claims 34-37, wherein the antifoam is simethicone.

39. The method of any one of claims 34-38, wherein the amount of antifoam at the initiation of increasing the culture volume is in the range of 23 mg / kg to 38 mg / kg.

40. The method of any one of claims 34-39, wherein the working volume at the initiation of the culture is 50% to 75% of the final working volume, the mammalian cells are CHO cells, the recombinant protein is an antibody, the antifoam is simethicone, and the amount of antifoam at the initiation of increasing the working volume is in the range of 23 mg / kg to 38 mg / kg.

41. A method for producing a recombinant protein comprising: initiating a culture in a bioreactor at a working volume that is at least 50% of a final working volume; inoculating the culture with mammalian cells engineered to express the recombinant protein; perfusing the culture for 24 hours to 72 hours at one or more perfusion rates of 0.05 V / d to 0.5 V / d, wherein foam coverage at the fluid surface of the bioreactor is maintained at 10% or less during the perfusing; increasing the working volume to the final working volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates, wherein at least one of the one or more feed rates is less than or equal to 0.50 V / d and at least one of the one or more permeate rates is less than or equal to 0.20 V / d, wherein each of the one or more feed rates is greater than the contemporaneous permeate rate and further wherein the bioreactor contains simethicone in an amount of at least 14 mg simethicone / kg of cell culture (mg / kg) at the initiation of increasing the working volume, wherein the amount of simethicone is calculated based on the final working volume; and once the final working volume is achieved, perfusing the culture at one or more perfusion rates of less than or equal to 0.5 V / d until the culture is terminated or harvested, wherein all rates expressed in V / d are calculated based on the final working volume.

42. The method of claim 41, wherein the working volume at the initiation of the culture is 50% to 75% of the final working volume.

43. The method of claim 41 or claim 42, wherein the mammalian cells are CHO cells.

44. The method of any one of claims 41-43, wherein the recombinant protein is an antibody.

45. The method of any one of claims 41-44, wherein the one or more perfusion rates during the 24 to 72 hour perfusing step are in the range of 0.10 V / d to 0.25 V / d.

46. The method of any one of claims 41-45, wherein the amount of simethicone at the initiation of increasing the culture volume is in the range of 23 mg / kg to 38 mg / kg.

47. The method of any one of claims 41-46, wherein the amount of simethicone in the bioreactor when the culture is terminated or harvested is between 80 mg / kg and 240 mg / kg.

48. The method of any one of claims 41-47, wherein the amount of simethicone in the bioreactor when the culture is terminated or harvested is between 100 mg / kg and 150 mg / kg.

49. The method of any one of claims 41-48, wherein the working volume at the initiation of the culture is 50% to 75% of the final working volume, the mammalian cells are CHO cells, the recombinant protein is an antibody, the one or more perfusion rates during the 24 to 72 hour perfusing step are in the range of 0. 10 V / d to 0.25 V / d, the amount of simethicone at the initiation of increasing the working volume is in the range of 23 mg / kg to 38 mg / kg, and the amount of simethicone in the bioreactor when the culture is terminated or harvested is between 100 mg / kg and 150 mg / kg.

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