Automatic bio production system, facility, and process

Abstract

To provide an automatic bio production system, facility, and process.SOLUTION: Disclosed are a process and an automated installation for producing target protein refined by using one or a plurality of disposable perfusion bioreactors, one or a plurality of disposable surge containers or one or a plurality of chromatography systems. The target protein may be recombinant or naturally occurring protein and / or therapeutic or other medically useful protein. For example, the disclosed process and automated installation are useful for producing a refined protein drug substance.SELECTED DRAWING: Figure 22

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 806,448, filed in the U.S. Patent and Trademark Office on February 15, 2019, which is incorporated by reference in its entirety.

[0002] The present invention relates to the field of automated manufacturing equipment and processes for the production of therapeutic proteins. [Background technology]

[0003] The biopharmaceutical industry is undergoing major change, driven in part by the rapid increase in approvals of new biotherapeutic drugs, increasing protein expression rates, and increasing pressures from the biosimilars market.

[0004] (Levine et al.,Efficient,flexible facilities for the 21st century,BioProcess International 10(11):20-30(2012))

[0005] The rapid increase in the pharmaceutical market share of biologics (from 11% in 2002 to approximately 20% in 2017), combined with the need for affordable access to medicines in developing regions of the world, necessitates the development of rapid, sustainable and cost-effective manufacturing methods.

[0006] (Walsh,Biopharmaceutical benchmarks 2014,Nature biotechnology 32(10):992-1002(2014))

[0007] As a result, there is a need to find alternative biopharmaceutical manufacturing technologies to replace traditional batch processing platforms to leverage key advantages such as higher throughput, operational flexibility and cost savings, and a reduced footprint with reduced environmental impact. Bioprocessing plants designed to include integrated upstream and downstream continuous manufacturing processes allow for rapid equipment changeover, product and capacity flexibility, and reduced manufacturing costs compared to batch culture processing. (For example, Farid et al.,Evaluating the economic and operational feasibility of continuous processes for monoclonal antibodies,Continuous Processing in Pharmaceutical Manufacturing pp.433-456(2015);Kelley,Industrialization of mAb production technology:the bioprocessing industry at a crossroads,mAbs 1(5):443-452(2009);Croughan et al.,The future of industrial bioprocessing: Batch or continuous?, Biotechnology and Bioengineering 112:648-651(2015); see Pollock et al., Fed-batch and perfusion culture processes: Economic, environmental, and operational feasibility under instability, Biotechnology and Bioengineering 110(1):206-219(2013)).

[0008] The advent of continuous perfusion technology has aided greater progress in connecting upstream process equipment to operate in continuous mode. This processing strategy has been valuable to several companies over the past 25 years, helping them overcome product-related stability issues. (Konstantinov et al.,White paper on continuous bioprocessing,Journal of Pharmaceutical Sciences 104(3):813-820(2015))

[0009] Modern cell lines and media have been engineered to target higher cell densities, especially when compared to fed-batch processing, with some cultures achieving viable cell densities of over 100 million cells / mL (Clincke et al., Very high density of chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in wave bioreactor™-part ii: Applications for antibody production and cryopreservation, Biotechnology Progress 29(3):768-777(2013)). As a result, the bottleneck in a typical biopharmaceutical manufacturing facility has shifted from the production bioreactor (upstream process) to the purification train (downstream process), especially the chromatography columns, due to their dimensional limitations. Purifying the large product batch sizes generated by the increasing amounts of protein from current production cell lines is not a trivial challenge. (Chon et al., Advances in the production and downstream processing of antibodies, New Biotechnology 28(5):458-463(2011)) Thus, the problem of integrating upstream biologics manufacturing processes with downstream processes continues to plague the biologics manufacturing industry.

[0010] Warikoo et al. reported integrating a continuous capture chromatography step downstream of the production bioreactor to reduce column size and buffer utilization. (Warikoo et al., Integrated continuous production of recombinant therapeutic proteins, Biotechnology and Bioengineering 109(12):3018-3029.(2012))

[0011] demonstrated that end-to-end continuous bioprocessing is feasible but still faces several challenges, including the development of robust viral clearance and automation strategies that ensure high product quality. (Godawat et al., End-to-end integrated fully continuous production of recombinant monoclonal antibodies, Journal of Biotechnology 213:13-19(2015))

[0012] The present invention provides a solution to these challenges and fills the need for an automated biopharmaceutical manufacturing technology that is an alternative to traditional batch processing platforms. Summary of the Invention

[0013] The present invention relates to an automated facility and method useful for producing purified proteins of interest, such as, but not limited to, therapeutic or other medically useful proteins. There are many challenges faced in maintaining long-term perfusion cultures with continuous capture of protein product. These include the large amount of medium consumed, and the large amount of fluid waste generated from the permeate before the start of product collection and the flow-through of the capture column during product collection. The need to maintain a sterile boundary in the waste line, the high cost of consumables and the labor can make collecting the waste in a closed bag system prohibitive. Long-term perfusion cultures increase the risk of contamination and have large sterile boundaries to maintain, all in the presence of rich growth medium, including during continuous capture operations. Other challenges include maintaining high viability cultures for extended periods of time and managing mismatched flow rates between connected unit operations, e.g., a perfusion bioreactor connected to a first chromatography system, a perfusion bioreactor connected to a viral inactivation system, a perfusion bioreactor connected to a second chromatography system, a perfusion bioreactor connected to an optional third chromatography system and / or viral filtration system, a perfusion bioreactor connected to an ultrafiltration / diafiltration system, etc.

[0014] The automated equipment and process of the present invention for producing purified proteins of interest (such as, but not limited to, therapeutic or other medically useful proteins) meets these and other objectives. In one aspect, the invention involves culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that allow the cells to secrete the protein into the liquid medium over a production culture period of at least 10 days, wherein during the production culture period, fresh sterile liquid medium is periodically or continuously added to the one or more disposable perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors directly related to a volume of culture that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed, and the volume of permeate removed is automatically and fluidly fed from the one or more disposable perfusion bioreactors to a disposable surge vessel and then to a first chromatography system, whereby the protein is collected in a protein isolate fraction.

[0015] In another aspect, the invention encompasses the use of multiple different concentrated media component solutions and aqueous diluents that are mixed simultaneously and delivered to the perfusion bioreactor as needed. In another aspect, the invention encompasses a closure process using gamma irradiated or autoclaved ready-to-use disposables, disposable sterile connectors, tube welders, and the use of chemical low-temperature sterilants on the columns. In another aspect, the invention encompasses efficient automated and regulated flow rates between fluidly connected and continuous unit operations, such as viral inactivation and various chromatography systems.

[0016] In one embodiment, the present invention relates to an automated facility for producing a purified protein of interest. The purified protein may be a recombinant protein or a naturally occurring protein. The automated facility is controlled by a process automation system (PAS) and includes:

[0017] (a) one or more disposable perfusion bioreactors capable of containing a liquid medium under conditions that permit the cultured cells to secrete protein into the liquid medium for a production culture period of at least 10 days, the disposable perfusion bioreactors being adapted to fluidly receive fresh sterile liquid medium into each of the perfusion bioreactors in direct relation to the volume of conditioned medium that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed during the production culture period;

[0018] (b) a first disposable surge vessel (SUSV1) into which the removed permeate volume is automatically and fluidly delivered from the one or more disposable perfusion bioreactors; and

[0019] (c) A first chromatography system adapted to automatically and fluidly receive the cell-free permeate from SUSV1, whereby proteins are captured in a protein isolate fraction.

[0020] The automated equipment of the present invention further comprises:

[0021] (d) a low pH or detergent viral inactivation system, and optionally a neutralization system, adapted to automatically and fluidly receive the protein isolate fraction from the first chromatography system, thereby obtaining a viral inactivation product pool comprising the protein; and

[0022] (e) a holding vessel or a second disposable surge vessel adapted to receive the viral inactivation product pool.

[0023] In some embodiments, the automated equipment may further include:

[0024] (f) a second chromatography system adapted to fluidly receive the viral inactivation product pool from the holding vessel or the second disposable surge vessel, thereby obtaining a purified product pool comprising the protein;

[0025] (g) an optional third chromatography system and / or virus filtration system adapted to fluidly receive the purified product pool comprising the protein from the second chromatography system, thereby obtaining a virus-free filtrate comprising the protein; and

[0026] (h) An ultrafiltration / diafiltration system adapted to fluidly receive the virus-free filtrate from the second chromatography system, or from the third chromatography system and / or the viral filtration system, thereby obtaining a purified protein of interest.

[0027] In some embodiments, the automated equipment for producing a purified protein of interest also includes a plurality of reservoirs, each adapted to contain a concentrated media component solution or an aqueous diluent, each of which is fluidly connected to the perfusion bioreactor either directly or indirectly through an optional mixing vessel adapted to receive and simultaneously mix the concentrated media component solution and the aqueous diluent in a predetermined ratio from the plurality of reservoirs, and the optional mixing vessel is fluidly connected directly to the perfusion bioreactor.

[0028] The present invention also relates to a method for producing a purified protein of interest, which may be a recombinant or naturally occurring protein, the process comprising the steps of:

[0029] (a) culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that permit the cells to secrete a protein into the liquid medium over a production culture period of at least 10 days, wherein during the production culture period, fresh sterile liquid medium that is simultaneously mixed from a plurality of different concentrated medium component solutions and an aqueous diluent is periodically or continuously added to the one or more disposable perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors in direct relation to a volume of culture that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed, and wherein the volume of permeate removed is automatically and fluidly delivered from the one or more disposable perfusion bioreactors to a disposable surge vessel and then to a first chromatography system, whereby the protein is collected in a protein isolate fraction.

[0030] The process of the present invention may further comprise the steps of:

[0031] (b) switching the protein isolate fraction to a low pH or detergent viral inactivation system, and optionally a neutralization system, to obtain a viral inactivation product pool containing the protein.

[0032] Additionally, the method may include the following additional polishing steps:

[0033] (c) introducing the viral inactivation product pool into a second chromatography system to obtain a purified product pool comprising the protein, wherein the viral inactivation product pool is introduced into the second chromatography system;

[0034] (d) switching the purified product pool containing the protein to an optional third chromatography system and / or a viral filtration system to obtain a virus-free filtrate containing the protein; and

[0035] (e) switching the virus-free filtrate through an ultrafiltration / diafiltration system to obtain a composition containing the purified protein of interest.

[0036] In a more particular aspect, the present invention relates to an automated facility for producing purified protein drug substance, i.e. purified protein of interest for therapeutic or other medical purposes (e.g. prophylactic or diagnostic purposes). The facility comprises:

[0037] (a) one or more disposable perfusion bioreactors capable of containing a liquid culture medium under conditions that permit cultured mammalian cells to secrete a protein of interest into the medium over a production culture period of at least 10 days, the disposable perfusion bioreactor being adapted to fluidly receive fresh sterile liquid culture medium into each of the perfusion bioreactors in direct relation to a volume of conditioned medium that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed during the production culture period, a plurality of reservoirs, each adapted to contain concentrated medium component solutions or aqueous diluents, are fluidly connected to the perfusion bioreactor directly or indirectly via an optional mixing vessel adapted to receive concentrated medium component solutions and aqueous diluents in predetermined ratios from the plurality of reservoirs and mix them simultaneously, the optional mixing vessel being fluidly connected directly to the perfusion bioreactor;

[0038] (b) a first disposable surge vessel (SUSV1), into which the removed permeate volume (cell-free) is automatically and fluidly supplied from one or more disposable perfusion bioreactors;

[0039] (c) a first chromatography system adapted to automatically and fluidly receive the cell-free permeate from SUSV1, whereby proteins are captured in a protein isolate fraction;

[0040] (d) a low pH or detergent viral inactivation system, and optionally a neutralization system, adapted to automatically and fluidly receive the protein isolate fraction from the first chromatography system, thereby obtaining a viral inactivation product pool comprising the protein;

[0041] (e) a holding vessel or disposable surge vessel adapted to receive the viral inactivation product pool;

[0042] (f) a second chromatography system adapted to fluidly receive the viral inactivation product pool from the holding vessel or the disposable surge vessel, thereby obtaining a purified product pool comprising the protein;

[0043] (g) an optional third chromatography system and / or virus filtration system adapted to fluidly receive the purified product pool comprising the protein from the second chromatography system, thereby obtaining a virus-free filtrate comprising the protein; and

[0044] (h) an UF / DF system adapted to fluidly receive the virus-free filtrate from the second chromatography system, or from the third chromatography system and / or the virus filtration system, thereby obtaining said purified protein drug substance. Operation of the automated equipment is controlled by a process automation system (PAS).

[0045] In another more particular aspect, the present invention relates to a process for producing a purified protein drug substance, i.e. a purified protein of interest for therapeutic or other medical purposes (e.g., prophylactic or diagnostic purposes). The purified protein drug substance may be a recombinant protein or a naturally occurring protein. The process comprises the following steps:

[0046] (a) culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that permit the cells to secrete a protein into the liquid medium over a production culture period of at least 10 days, wherein during the production culture period, fresh sterile liquid medium is periodically or continuously added to the one or more disposable perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors in direct relation to a volume of culture that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed, and the volume of permeate removed is automatically and fluidly delivered from the one or more disposable perfusion bioreactors to a disposable surge vessel and then to a first chromatography system, whereby said protein is collected in a protein isolate fraction;

[0047] (b) switching the protein isolate fraction to a low pH or detergent viral inactivation system, and optionally a neutralization system, to obtain a viral inactivation product pool containing the protein;

[0048] (c) introducing the viral inactivation product pool into a second chromatography system to obtain a purified product pool comprising the protein;

[0049] (d) switching the purified product pool containing the protein to an optional third chromatography system and / or a viral filtration system to obtain a virus-free filtrate containing the protein; and

[0050] (e) Switching the virus-free filtrate into an ultrafiltration / diafiltration system to obtain purified drug substance containing the protein of interest.

[0051] In some embodiments of the process for producing purified protein drug substance, fresh sterile liquid medium is mixed simultaneously from multiple different concentrated medium component solutions and aqueous diluents and then added to one or more perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors. The foregoing summary is not intended to define all aspects of the invention, and additional aspects are described in other sections, such as the Detailed Description of the Embodiments. It is understood that the entire document is intended to be linked as a unified disclosure, and all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of this document.

[0052] In addition to the above, the present invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations defined by the specific paragraphs above. For example, a particular aspect of the invention described as a genus should be understood to mean that every member of the genus is an aspect of the invention individually. Also, an aspect described as a genus or selecting a member of a genus should be understood to encompass combinations of two or more members of that genus. Although the applicant has invented the full scope of the invention described herein, the applicant does not intend to claim subject matter described in other prior art documents. Thus, if statutory prior art within the scope of a claim is brought to the applicant's attention by the patent office or other entity or individual, the applicant reserves the right to exercise amendment rights under applicable patent law to redefine the subject matter of such claim to specifically exclude such statutory prior art or obvious variations of the statutory prior art from the scope of such claim. Variations of the invention defined by such amended claims are also contemplated as aspects of the present invention. [Brief description of the drawings]

[0053] [Figure 1A]FIG. 1 shows a schematic partial process flow diagram of one embodiment of the process of the present invention showing multiple disposable reservoirs fluidly connected to a 500L scale disposable perfusion bioreactor (herein referred to as "500L SUB"), each reservoir holding a different sterile concentrated media component (the reservoirs shown are shown here with their contents "50% Glucose", "Cys / Tyr Stock", and "600L Tote 7.5x Concentrate", respectively) or aqueous diluent ("1 kL Tote WFI"). "AF" = antifoam agent used to minimize foaming in the bioreactor, "Base" = sodium carbonate added by automation to maintain bioreactor pH.

[0054] [Figure 1B]A schematic partial process flow diagram of a semi-continuous embodiment of the process of the present invention is shown, from a 500 L disposable bioreactor ("SUB" and "batch unit (A)"), to a perfusion system ("perfusion skid" and "batch unit (A1)"), to a disposable surge vessel (SUSV, "non-batch unit (B1)" labeled "200 L portable mixer"), to a simulated moving bed (SMB) first chromatography system ("batch unit (B)", represented here as a disposable multi-column chromatography system on a cart labeled "SMB Chrom System"), to an elution collection vessel for collecting protein isolate fractions ("non-batch unit (B3)" and "non-batch unit (B4)" labeled "100 L portable mixer"). In the embodiment shown in FIG. 1B, upstream of the SUSV, an optional filtration system ("non-batch unit (B2)" labeled "filter bank") protects the first chromatographic system from particulates, and an optional heat exchanger cools the permeate material to room temperature (RT), or in some embodiments to 4° C. or another desired temperature, prior to introduction into the first chromatographic system, depending on the needs of the chromatographic system's components and protein molecule stability. In other embodiments (not shown here in FIG. 1B), the protein isolate fraction can be fluidly fed to a second disposable surge vessel (SUSV2), or to at least two automatically switchable alternative disposable collection vessels (SUCV1 and SUCV2), or directly and continuously to a viral inactivation system (e.g., a low pH or detergent viral inactivation system). In the schematic of the embodiment shown here in Figure 1B, a disposable air break assembly (see also Figure 2) is used to send the permeate to waste (downstream of the SUB and perfusion skid) at the start of the perfusion run before the first chromatography system and to drain the flow-through waste downstream of the first chromatography system. An additional unit operation between the perfusion bank and the filter bank (as shown in Figure 1B) can optionally be included for single pass tangential flow filtration (SPTFF) to concentrate the perfusion permeate before it flows further downstream towards the first chromatography system.

[0055] [Figure 1C] A schematic partial process flow diagram of one embodiment of the process of the present invention is shown in which two portable mixers (here shown as 500 L volume each) function as alternating SUCV1 and SUCV2, respectively, operating as part of or feeding into a viral inactivation system ("viral inactivation skid"), different embodiments are also shown diagrammatically in Figures 20A, 20C, and 20D. In this Figure 1C, the SUSV is shown as a 500 L portable mixer large enough to accommodate the entire pool. Alternatively, in a continuous or semi-continuous operation embodiment, any of the SUSVs shown (i.e., SUSV2, SUSV3, or SUSV4) may instead be of a different convenient volume (e.g., 50 L, 75 L, or 100 L) or arbitrary. For example, the disposable surge receptacle shown in front of the UF / DF system skid (i.e., SUSV4) can optionally be eliminated in order to use the UF / DF skid recirculation tank as the surge receptacle instead (see, e.g., FIG. 20E). In other embodiments, the disposable surge receptacles between the second and third chromatography systems (i.e., SUSV2) or between the second and viral filtration systems (i.e., SUSV2) can be eliminated in order to run two or more purification or "polishing" steps in parallel (see, e.g., FIG. 20F).

[0056] [Figure 1D]FIG. 1D shows a schematic partial process flow diagram of one embodiment of the process of the present invention, in which two portable mixers (each 500 L volume) function as alternating SUCV1 and SUCV2, respectively, operating as part of or feeding into a viral inactivation system ("viral inactivation skid"). In the embodiment shown in FIG. 1D, downstream of the viral inactivation system (e.g., low pH viral inactivation system and neutralization system) and depth filtration, the process proceeds in batch mode with holding vessels between steps or operations (shown as HV1, HV2, HV3, and HV4). However, in other embodiments, any of HV2, HV3, or HV4 can be replaced by a surge vessel or eliminated entirely for uninterrupted flow between steps or operations under automated control.

[0057] [Diagram 2] 2 is a schematic diagram of one embodiment of a disposable air break assembly. Shown are (A) the connection to the waste outlet line of the system, (B) a vent filter to introduce an air break into the flowing liquid, (C) larger and smaller tubing sections to maintain the air break, and (D) the connection to a drain. The cross-hatched tubing in FIG. 2 (e.g., C) represents braided tubing, although other non-braided tubing of appropriate diameter could be used instead.

[0058] [Diagram 3] FIGURE 3 is a schematic diagram of one embodiment of a SUSV1 ("SUSV") volume control. The volume restrictions shown in FIGURE 3 at which control action occurs are merely exemplary and may vary based on the volume and flow capacity of system components.

[0059] [Figure 4]FIG. 4 shows a schematic partial process set-up for one embodiment of sanitizing chromatography resins and column housings with a suitable chemical sanitizer, e.g., peracetic acid (indicated as "PAA" in FIG. 4). In this embodiment, a disposable multi-column continuous chromatography system, e.g., a simulated moving bed (SMB) chromatography system, is referred to herein as "BioSMB" to represent the Cadence™ BioSMB® PD System (Pall Life Sciences), although other suitable disposable multi-column continuous capture chromatography systems can be substituted. In this embodiment, the "sterile connector A" can be an AseptiQuik® G connector (Colder Products Company), or the like, the "sterile connector B" can be a Kleenpak® Genderless Connector (Pall Biotech), or the like, the tubing can be size 73 silicone tubing, or the like, and the closed bag for waste can be a 10 L disposable bag, or another convenient volume.

[0060] [Diagram 5] Schematically illustrates various hardware and software components of an embodiment of an automated equipment of the present invention for producing a purified protein of interest, such as a purified protein drug substance, that allows for communication of data between different components of the system. However, each of the components shown in grey boxes of the perfusion system and continuous chromatography system skid, namely, filter bank, feed tank A and feed tank B, and harvest tank A and harvest tank B, are entirely optional as components of such a skid (batch unit). In other embodiments of the present invention, any of these components may be present instead in a non-batch unit configuration, or may not be present, as desired for a particular manufacturing purpose.

[0061] [Figure 6]5 shows the viable cell densities of the 500 L bioreactor runs and the corresponding 2 L comparator satellite bioreactors designated "Run 1 R17," "Run 2 R14," and "Run 3 R21," respectively.

[0062] [Figure 7] 500L bioreactor run and corresponding 2L satellite bioreactor viability are shown.

[0063] [Figure 8] 1 shows the cell bleed rates of the 500 L bioreactor run and the corresponding 2 L satellite bioreactor.

[0064] [Figure 9] Figure 1 shows the permeate productivity of the 500 L bioreactor run and the corresponding 2 L satellite bioreactor.

[0065] [Figure 10] Culture volume control of a 500 L disposable bioreactor (SUB) using water for injection (WFI) as needed. SUB levels (right scale, top plot) are shown in kilograms and WFI time flow rates (left scale, bottom plot and steps) are shown in mL / min. Step changes in WFI flow rate correspond to increasing perfusion rates from 0.5 to 1.0 to ~2.0 vvd.

[0066] [Figure 11] Representative data comparing osmolality in a 500 L SUB with a 2 L satellite is shown (Run 3 shown).

[0067] [Figure 12] Representative data comparing CO2 of a 500L SUB to a 2L satellite is shown (Run 3 shown).

[0068] [Figure 13]Representative data comparing base usage in a 500L SUB to a 2L satellite is shown (Run 3). Data for the 500L SUB are shown as base usage in mL / day normalized to the working volume of the 2L bioreactor (1.5L culture volume).

[0069] [Figure 14] Representative data comparing lactate specific production rates of the 500L SUB and the 2L satellite are shown (run 3 shown).

[0070] [Figure 15] Representative data comparing the specific glucose consumption rate of a 500 L disposable perfusion bioreactor with a 2 L satellite is shown ("Run 3 R21" and "Run 3 R22").

[0071] [Figure 16A] Representative elution profiles of absorbance at 280 nm (A280, Y-axis) of Protein A affinity chromatography for each of three separate Protein A columns (designated "Col1," "Col2," and "Col3" in Figures 16A-B) on a BioSMB (shown as one elution cycle per day for 17 days, minutes, Figure 16A) and their respective elution column volumes (CV, Figure 16B) per elution cycle (Run 2 shown). [Figure 16B]Representative elution profiles of absorbance at 280 nm (A280, Y-axis) of Protein A affinity chromatography for each of three separate Protein A columns (designated as "Col1," "Col2," and "Col3" in Figures 16A-B) on BioSMB (shown as one elution cycle per day for 17 days, minutes, Figure 16A) and their respective elution column volumes (CV, Figure 16B) per elution cycle (run 2 shown). In Figure 16B, the thick solid plot line represents Col1 data (middle plot on day 17), the thin solid plot line represents Col3 data (top plot on day 17), and the hatched plot line represents Col2 data (bottom plot on day 17).

[0072] [Figure 17] Representative Protein A step yield data (elution yields) are shown, shown as the combined daily pool of elution cycles (Run 2 shown). The cumulative elution cycles ("Cumulative Elution Cycles") are also shown.

[0073] [Figure 18] Representative process-related impurities in the combined daily neutralized elution pools are shown (Run 2 shown). HCP = host cell protein as measured by ELISA assay, DNA = host cell DNA as measured by qPCR assay, LPrA = leached protein A as measured by ELISA assay.

[0074] [Figure 19A] Representative SUSV1 culture volume control and multi-column continuous capture simulated moving bed (SMB) primary chromatography system (referred to herein as "BioSMB") loading flow rates (run 2 shown). [Figure 19B]Representative SUSV1 culture volume control and multi-column continuous capture simulated moving bed (SMB) first chromatography system (herein referred to as "BioSMB") load flow rates (run 2 shown). In Figure 19A, the load flow rate (right y-axis scale, L / time) is shown in the top plot and the pressure (left y-axis scale, bar) is shown in the jagged bottom plot, where the flow rate was varied ±10% to maintain the SUSV control range. In Figure 19B, the volume (measured as weight, kg) contained in the disposable surge vessel (SUSV) is shown, with a set point of 100 kg and a control range of 70 kg to 130 kg, with an assumed density of 1 kg / L.

[0075] [Figure 20A] A schematic partial process flow diagram of one embodiment of the process of the present invention is shown in which two alternating disposable collection vessels (SUCV1 and SUCV2) are operated in an alternating manual batch format as the structure in which viral inactivation (and optionally neutralization) is performed. For example, acidification and neutralization can be performed alternately in SUCV1 and SUCV2.

[0076] [Figure 20B] FIG. 1 shows a schematic partial process flow diagram of one embodiment of the process of the present invention, in which a disposable surge vessel (shown as SUSV2) is interposed between a first chromatography system, such as a simulated moving bed (SMB) chromatography system (called "BioSMB"), and a viral inactivation / neutralization skid that includes a viral inactivation system and a neutralization system in an uninterrupted flow or continuous format. In another continuous flow embodiment, SUSV2 may be the vessel in which viral inactivation (and optionally neutralization) occurs.

[0077] [Figure 20C]FIG. 20C shows a schematic partial process flow diagram of one embodiment of the process of the present invention in which two alternating disposable collection vessels (SUCV1 and SUCV2) feed a viral inactivation / neutralization skid containing a viral inactivation ("VI") system and a neutralization ("Neut") system in a batch format. The viral inactivation / neutralization system can consist of a single tank or two alternating tanks. In the embodiment shown in FIG. 20C, upstream of the SUSV (SUSV1), an optional ("Optional") filter bank protects the first chromatographic system from particulates, and an optional heat exchanger ("Heat Exchanger (Optional)") cools the permeate material to room temperature (RT), or in some embodiments, 4° C. or another desired temperature, depending on the stability needs of the components and protein molecules of the chromatographic system, before introduction into the first chromatographic system (referred to as "BioSMB").

[0078] [Figure 20D] FIG. 1 shows a schematic partial process flow diagram of one embodiment of the process of the present invention in which two alternating disposable collection vessels (SUCV1 and SUCV2) operate as part of, rather than simply feeding into, a viral inactivation / neutralization skid that includes an automated batch-style viral inactivation and neutralization system. Acidification and neutralization processes alternate in the SUCV1 and SUCV2 tanks.

[0079] [Figure 20E]20E shows a schematic partial process flow diagram of one embodiment of the process of the present invention in an uninterrupted or continuous flow format. In this embodiment, a disposable surge receptacle (shown as "receptacle") is located between the process steps / operations, for example, the second chromatography system (shown as "chrome 2"), the optional third chromatography system (shown as "chrome 3"), the virus filtration system (shown as "VF"), and optionally ("*optional"), the ultrafiltration / diafiltration ("UF / DF") system, since the recirculation tank of the UF / DF skid can be used as a surge receptacle instead. As shown in FIG. 20E, optional ("optional") in-line adjustment of pH and / or adjustment of conductivity loading of the effluent from each operation can be automatically performed before the next operation, if necessary. In other alternative embodiments, adjustment of pH and / or adjustment of conductivity loading of the effluent from each operation can be performed in one or more of the "receptacles" or SUSVs (i.e., in-vessel adjustments) shown between the operations in FIG. 20E.

[0080] [Figure 20F] A schematic partial process flow diagram of one embodiment of the process of the present invention is shown in the form of an uninterrupted or continuous flow in which two or more steps / operations are performed in tandem without an intervening disposable surge receptacle, for example (in FIG. 20F) a second chromatography system (shown as "Chrome 2"), an optional third chromatography system (shown as "Chrome 3"), and / or a viral filtration system (shown as "VF"), or for example (in FIG. 20G) upstream of a viral filtration system (shown as "VF"), in-line depth filtration (shown in FIG. 20G as "in-line DF", also known as ILDF) and single pass tangential flow filtration (shown as "SPTFF"). [Figure 20G]A schematic partial process flow diagram of one embodiment of the process of the present invention is shown in the form of an uninterrupted or continuous flow, in which two or more steps / operations are performed in tandem without an intervening disposable surge vessel, for example (in FIG. 20F) a second chromatography system (shown as "Chrome 2"), an optional third chromatography system (shown as "Chrome 3"), and / or a virus filtration system (shown as "VF"), or for example (in FIG. 20G) upstream of a virus filtration system (shown as "VF"), in-line depth filtration (shown as "in-line DF" in FIG. 20G, also known as ILDF) and single pass tangential flow filtration (shown as "SPTFF"). From the single pass tangential flow filtration, the flow can continue to UF / DF or can be collected in a holding vessel for batch application of UF / DF. When the process of the present invention involves switching the virus-free filtrate into an UF / DF system to obtain a composition comprising a purified protein of interest, it is preferred that the operating pressure of the SPTFF step is controlled in the range of about 0.25 psi to about 60 psi (or about 0.25 psi to about 45 psi, or about 0.25 psi to about 30 psi, or about 0.25 psi to about 15 psi, or about 0.25 psi to about 5 psi) and / or the operating pressure of the ILDF step is controlled in the range of about 0.25 psi to about 60 psi (or about 0.25 psi to about 45 psi, or about 0.25 psi to about 30 psi, or about 0.25 psi to about 15 psi, or about 0.25 psi to about 5 psi).

[0081] [Figure 21] 1 shows a comparison of high molecular weight species (HMW) as measured by size-exclusion high performance liquid chromatography (SE-HPLC) between the protein isolate fraction after Protein A chromatography ("PrAEL HMW") and the low pH viral inactivation and neutralized viral inactivation product pool ("VI / neutralized pool HMW").

[0082] [Figure 22]FIG. 1 shows a schematic diagram of one embodiment of the continuous process of the present invention for producing a purified protein of interest or purified protein drug substance from a single-use perfusion bioreactor ("SUB") to a final formulation step including a two-stage single-pass tangential flow filtration ("SPTFF") and in-line diafiltration ("ILDF") module. The first chromatography system was a Protein A affinity chromatography capture step performed using a Cadence™ BioSMB® PD system (Pall, referred to as "BioSMB"), a low pH viral inactivation system ("2-Tank VI") was included in the process, and the second chromatography system included ion exchange chromatography ("IEX"). A single-use surge vessel ("SUSV") is shown used during unit operation.

[0083] [Figure 23] FIG. 23 is a schematic diagram of an example depth filter cart and its post-use water-washing system. The cart in this embodiment was constructed with two filter trains, each with a depth filter (herein referred to as "DF-1", "DF-2") followed by a sterile filter (herein referred to as "SF-1" and "SF-2", respectively). Differential pressure was monitored across each filter using pressure transducers (herein referred to as "P1", "P2", "P3", and "P4"). At designated differential pressure limits, the filter trains can be switched using automated valves (two triangles pointing towards each other with the tips of the inner points touching represent two-way valves). Dirty filters can be replaced and washed for later reuse while the new filters are in operation.

[0084] [Figure 24]FIG. 1 is a detailed schematic diagram of an SPTFF and ILDF system in an exemplary continuous format embodiment, as described in Example 5. Differential pressure was monitored across each filter using pressure transducers (herein referred to as "P1", "P2", "P3", and "P4"). An optional "break tank" denotes an optional surge vessel. At designated differential pressure limits, filter trains can be switched using automated valves (two triangles pointing to each other with inner points touching represent two-way valves, and three triangles pointing to each other with inner points touching represent three-way valves). Dirty filters can be replaced and cleaned for later reuse while new filters are in operation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0086] definition

[0087] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art. Further, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Thus, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a reference to a "protein" includes one protein or multiple proteins, and a reference to a "bioreactor" includes one bioreactor or multiple bioreactors.

[0088] The present invention relates to an integrated, continuous or semi-continuous, automated process for producing purified proteins of interest (such as, but not limited to, purified protein drug substances). The process of the invention is carried out under sterile operating conditions and includes automated control adjustment of flow rates of a chromatography system. (See, e.g., FIG. 5).

[0089] The various steps of the process can be carried out in automated facilities, either in a single clean room or in multiple separate modular clean rooms, and can optionally be automated and controlled.

[0090] The term "integrated" in the context of a process for manufacturing a purified protein of interest (such as, but not limited to, a protein drug substance) means that one or more upstream and / or downstream steps of the manufacturing process are performed under common or coordinated control based on programmed commands modified by current sensor feedback of defined parameters related to set points or flow rates. The term "coordinated" means that two or more operations, steps, processes, components, or systems are controlled, coordinated, or scheduled in a relationship that ensures efficiency or harmony of their functioning toward a single purpose.

[0091] "Upstream" processes include, but are not limited to, for example, culturing recombinant host cells, removing cells from the permeate, and fluidizing a volume of cell-free permeate from one or more perfusion bioreactors to a disposable surge vessel. "Downstream" process steps include, but are not limited to, for example, capture and purification of the product in a first chromatography system, switching a protein isolate fraction to a virus inactivation system that is a low pH or detergent virus inactivation system and optionally (e.g., in embodiments of a low pH virus inactivation system) a neutralization system, introducing a virus inactivation product pool to a second chromatography system, switching a purified product pool to a third chromatography system and / or a virus filtration system, and / or switching a virus-free filtrate to an ultrafiltration / diafiltration system.

[0092] A "viral inactivation product pool" includes protein product-containing material obtained by operation of a viral inactivation system (and optionally a neutralization system). For purposes of the present invention, a "viral inactivation product pool" encompasses such material obtained by operation of a viral inactivation system (and optionally a neutralization system) and then filtered by (optional) depth filtration to obtain a filtered viral inactivation product pool (FVIP) prior to further downstream processing.

[0093] A "continuous" form of manufacturing process or system refers to a processing mode in which a perfusion bioreactor is fluidly connected to a continuous capture chromatography step (e.g., processing with a first chromatography system) with uninterrupted flow coming from the bioreactor (directly or indirectly through an intervening unit operation) to a first chromatography system, followed by a downstream viral inactivation step, and optionally from the uninterrupted flow to depth filtration. Further downstream product purification steps (e.g., processing with a second chromatography system, an optional third chromatography system, viral filtration, and UF / DF) are fluidly connected to each other in series, with optional intervening surge vessels, all with uninterrupted flow to the aforementioned upstream processing steps.

[0094] A "semi-continuous" form of production process refers to a processing regime in which the perfusion bioreactor is fluidly connected with uninterrupted flow to a continuous capture chromatography step (e.g., processing with a first chromatography system), and to processing with a viral inactivation system, and optionally with uninterrupted flow to depth filtration and storage of the viral inactivation product pool in a holding vessel (HV1). Temporary storage of the viral inactivation product pool in the holding vessel is followed by one or more batch downstream processing steps, which can be fluidly connected to each other in succession with uninterrupted flow, for example, processing with ultrafiltration / diafiltration with a second chromatography system, an optional third chromatography system, and possibly any intervening surge or holding vessels (i.e., holding vessels, if there are more than one batch process or operation).

[0095] A "perfusion bioreactor" is a bioreactor for culturing cells, in which equal volumes of medium can be added and removed from the reactor while the cells are retained in the bioreactor. A perfusion bioreactor includes a bioreactor and an operably attached perfusion system that provides a steady source of fresh nutrient medium and removal of cellular waste products. The bioreactor and the perfusion system of the perfusion bioreactor may be separate mechanical units that operate in concert. Numerous commercially available examples include, but are not limited to, various Xcellerex® brand disposable bioreactors (SUB, GE Healthcare Life Sciences) and KrosFlo® brand perfusion channel assemblies and systems (Spectrum, Repligen), which bioreactors and perfusion systems can be appropriately combined into a perfusion bioreactor by one of skill in the art. Alternatively, the bioreactor and perfusion system can be assembled into a single mechanical unit, such as, but not limited to, a 3D Biotek brand perfusion bioreactor (Sigma-Aldrich). The secreted protein product in the bioreactor can be continuously collected by microfiltration during the process of removing the medium through the perfusion system, and thus the protein of interest is isolated in the microfilter permeate exiting the perfusion system.

[0096] A manufacturing process or system step in an automated manufacturing facility is "fluidically" performed, "fluidically connected" to, or "fluidically" receives material from another step of the manufacturing process or another system when material containing the protein of interest flows by pipes, tubes, or other closed conduits between the steps or systems without manual loading or unloading. A manufacturing process or system step in an automated manufacturing facility is generally referred to as a "unit operation." A unit operation configured to communicate (e.g., by hardwired or wireless connection) with an OPC server is referred to as a "batch unit" or "skid." Typically, but not necessarily, the disposable bioreactor, the perfusion system, the first chromatography system, the second chromatography system, the optional third chromatography system, and the ultrafiltration / diafiltration system are configured as "skids." (See, e.g., FIGS. 1B-1D.) The viral inactivation system, and optionally the neutralization system, may be skids in some embodiments. (See, e.g., FIG. 20D.) Unit operations that are controlled not via hard wiring, but via dongles and / or Profibus devices, or similar digital information storage devices and electronic hardware connectors, are referred to as "non-batch units." For convenience and flexibility, filter banks, heat exchangers, surge receptacles, supply tanks, reservoirs, holding vessels, collection vessels or harvesting tanks (e.g., elution collection vessels), and portable mixers and other mixing vessels, when present as needed, are typically configured as non-batch units, although in some embodiments unit operations such as these may also be included in a "skid" that includes control via hard wiring or wireless connections. (See, e.g., FIG. 5 and FIG. 20D.)

[0097] The terms "automated," "automation-controlled," or "automatically," used interchangeably in reference to a manufacturing process or facility, refer to computer control of the performance or execution of one or more process steps or the operation of a component or system of the manufacturing facility, possibly with accompanying feedback control of the process steps or operations. Typically, a computerized controller receives digital signals from a detector of the physical or chemical parameter being controlled and issues responsive digital instructions to a system or subsystem.

[0098] The term "therapeutic protein" refers to a pharmacologically active protein applicable for the prevention, treatment, or cure of a human disease or condition. Examples of therapeutic proteins include, but are not limited to, monoclonal antibodies, recombinant forms of natural proteins (e.g., receptors, ligands, hormones, enzymes, or cytokines), fusion proteins, peptibodies, and / or monomer domain binding proteins based on domains selected from, for example, the LDL receptor A domain, the thrombospondin domain, the thyroglobulin domain, the trefoil / PD domain, the VEGF binding domain, the EGF domain, the anato domain, the notch / LNR domain, the DSL domain, the integrin beta domain, and the Ca-EGF domain. The above are merely exemplary, and therapeutic proteins can include any clinically relevant polypeptide targeting moiety or polypeptide ligand. The term "derivative", when used in connection with a therapeutic protein of interest, refers to proteins that have been covalently modified by conjugation to therapeutic or diagnostic agents, labeling (e.g., with radionuclides or various enzymes), covalent attachment of polymers such as PEGylation (derivatization with polyethylene glycol), and insertion or substitution of natural or non-natural amino acids.

[0099] A "drug substance" is an active pharmaceutical ingredient (API) intended to provide pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the body. The drug substance may be further formulated or reformulated with buffers, carriers, and / or excipients, and the drug substance may be further administered in a dosage form suitable and / or approved for clinical use.

[0100] The term "purifying" a desired protein or "purification" refers to increasing the purity of a desired protein from a composition or solution that contains a protein of interest (i.e., a "POI", e.g., a therapeutic or other medically useful protein) and one or more contaminants by removing (completely or partially) at least one contaminant from the composition or solution. An "isolated" protein is one that has been identified and separated from one or more components of its natural environment or the medium secreted by the producing cell. In some embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural or medium environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use. "Contaminant" components of its natural environment or medium are materials that would interfere with the industrial, research, therapeutic, prophylactic or diagnostic use of the protein of interest and may include enzymes, hormones, and other proteinaceous or non-proteinaceous (e.g., polynucleotides, lipids, carbohydrates) solutes. Typically, an "isolated protein" or, interchangeably, a "protein isolate" comprises at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. In some embodiments, an isolated protein of interest is "purified" (1) to greater than 95% by weight of the protein, most preferably greater than 99% by weight, or (2) to homogeneity by SDS-PAGE or other suitable techniques under reducing or non-reducing conditions, optionally using a stain, such as Coomassie blue or silver stain. An isolated naturally occurring antibody includes an antibody in situ within a recombinant cell, since at least one component of the protein's natural environment will not be present. Typically, however, an isolated or purified protein of interest (e.g., a purified protein drug substance) is prepared by at least one purification step.

[0101] Proteins of interest for purposes of the present invention, such as therapeutic or other medically useful proteins, whether or not they contain variant or parent antibody amino acid sequences, are typically produced by recombinant expression techniques, but may also be naturally occurring proteins.

[0102] "Polypeptide" and "protein" are used interchangeably herein and include a molecular chain of two or more amino acids covalently linked via peptide bonds. These terms do not refer to a specific length of the product. Thus, "peptide" and "oligopeptide" are included within the definition of a polypeptide. The term includes post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Additionally, protein fragments, analogs, mutant or variant proteins, fusion proteins, and the like, are included within the meaning of a polypeptide. The term also includes molecules in which one or more amino acid analogs or non-standard or non-natural amino acids are included, such that they can be recombinantly expressed using known protein engineering techniques. Additionally, proteins can be derivatized as described herein and by other known organic chemistry techniques.

[0103] The term peptide or protein "analog" refers to a polypeptide having a sequence that differs from a naturally occurring peptide sequence by at least one amino acid residue substitution, internal addition, or internal deletion of at least one amino acid, and / or amino- or carboxy-terminal truncation or addition. An "internal deletion" refers to the absence of an amino acid from the naturally occurring sequence at a position other than the N- or C-terminus. Similarly, an "internal addition" refers to the presence of an amino acid in the naturally occurring sequence at a position other than the N- or C-terminus.

[0104] A "variant" of a polypeptide (e.g., an immunoglobulin, or antibody, or a fusion protein) comprises an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted in the amino acid sequence compared to another polypeptide reference sequence. Variants can include variants of fusion proteins.

[0105] The term "fusion protein" indicates that a protein contains polypeptide components derived from two or more parent proteins or polypeptides. Typically, a fusion protein is expressed from a "fusion gene" in which a nucleotide sequence encoding a polypeptide sequence from one protein is added in frame to a nucleotide sequence encoding a polypeptide sequence from a different protein, optionally separated by a linker. The fusion gene can then be expressed by a recombinant host cell as a single protein. Fusion proteins incorporating antibodies or antigen-binding portions thereof are known.

[0106] The process of the invention involves culturing mammalian cells, e.g., recombinant host cells, capable of producing a secreted protein of interest. A "secreted" protein refers to a protein that can be directed to the endoplasmic reticulum (ER), secretory vesicles, or extracellular space as a result of a secretory signal peptide sequence, as well as a protein that is released into the extracellular space without necessarily including a signal sequence. Once a secreted protein is released into the extracellular space, it can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by a number of mechanisms, including exocytosis and proteolytic cleavage. In some other embodiments, the antibody protein of interest can be synthesized by the host cell as a secreted protein and then further purified from the extracellular space and / or medium.

[0107] As used herein with respect to proteins produced by recombinant DNA technology in host cells, "soluble" is a protein that is present in an aqueous solution; if the protein contains a twin-arginine signal amino acid sequence, the soluble protein is excreted into the periplasmic space in a gram-negative bacterial host, or secreted into the medium by eukaryotic host cells with secretory capabilities (i.e., "protein-secreting" cells, e.g., protein-secreting mammalian cells), or by bacterial hosts with appropriate genes (e.g., the kil gene). Thus, a soluble protein is one that is not found in inclusion bodies within the host cell. Alternatively, depending on the context, a soluble protein is one that is not found incorporated into cell membranes or that can be dissolved in vitro or dissolved in an aqueous buffer without forming a significant amount of insoluble aggregates under physiological conditions (i.e., less than 10% of the total protein forms aggregates, typically less than about 5%) when suspended without other proteins in an aqueous buffer of interest under physiological conditions, e.g., a buffer that does not contain an ionic detergent or chaotropic agent such as sodium dodecyl sulfate (SDS), urea, guanidinium hydrochloride, or lithium perchlorate. In contrast, an insoluble protein is one that exists in a denatured form inside cytoplasmic granules (called inclusion bodies) in a host cell, or, similarly depending on the context, an insoluble protein is one that exists in a cellular membrane, including but not limited to the cytoplasmic membrane, mitochondrial membrane, chloroplast membrane, endoplasmic reticulum membrane, etc., or is a protein that forms a significant amount of insoluble aggregates in in vitro aqueous buffers under physiological conditions (i.e., forms aggregates that are about 10% or more of the total protein) when suspended without other proteins (at a physiologically compatible temperature) in an aqueous buffer of interest under physiological conditions, e.g., a buffer that does not contain an ionic detergent or chaotropic agent such as sodium dodecyl sulfate (SDS), urea, guanidinium hydrochloride, or lithium perchlorate.

[0108] A "stable" formulation of a protein is one in which the protein essentially retains its physical and / or chemical stability and / or biological activity upon processing (e.g., ultrafiltration, diafiltration, other filtration steps, vial filling), shipping and / or storage of the antibody drug substance and / or drug product. Together, the physical, chemical and biological stability of the protein in the formulation embody the "stability" of the protein formulation specific to the conditions under which the formulated drug product (DP) is stored. For example, a formulation stored at sub-zero temperatures is expected to have no significant changes in either chemical, physical or biological activity, while a formulation stored at 40°C is expected to have changes in its physical, chemical and biological activity, with the extent of the changes depending on the storage time of the drug substance or drug product. The composition of the protein formulation can also affect the rate of change. For example, aggregate formation is greatly affected by protein concentration, with higher aggregation rates observed at higher protein concentrations. Excipients are also known to affect the stability of formulations, for example, by the addition of salts, which increase the aggregation rate of some proteins, while other excipients, such as sucrose, are known to decrease the aggregation rate during storage. Instability is also greatly affected by pH, resulting in both higher and lower degradation rates, depending on the type of modification and the pH dependency.

[0109] A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Wang, W. (1999), Instability, stabilization and formulation of liquid protein pharmaceuticals, Int J Pharm 185:129-188. Stability can be measured at a selected temperature for a selected period of time. For rapid screening, for example, the formulation may be kept at 40°C for 2 weeks to 1 month, at which point stability is measured. If the formulation is stored at 2-8°C, generally the formulation should be stable at 30°C for at least 1 month, or at least 1 week at 40°C, and / or at least 2 years at 2-8°C.

[0110] A protein "retains its physical stability" in a formulation if it shows minimal signs of changes to secondary and / or tertiary structure (i.e., native structure), or aggregation, and / or precipitation and / or denaturation, as measured by visual inspection of color and / or clarity, or by UV light scattering or size exclusion chromatography or other suitable methods. Protein physical instability, i.e., loss of physical stability, can be caused by oligomerization, resulting in dimers and higher order aggregates, subvisible and visible particle formation, and precipitation. The extent of physical degradation can be confirmed using various techniques depending on the type of degradant of interest. Dimers and higher order soluble aggregates can be quantified using size exclusion chromatography, while subvisible particles can be quantified using light scattering, light obscuration, or other suitable techniques.

[0111] A protein "retains its chemical stability" in a formulation if the chemical stability at a given time is such that no covalent bonds are formed or broken, resulting in a change in the primary structure of the protein components. The change in the primary structure may result in modification of the secondary and / or tertiary and / or quaternary structure of the protein, leading to the formation of aggregates or reversal of already formed aggregates. Exemplary chemical modifications include isomerization, deamidation, N-terminal cyclization, backbone hydrolysis, methionine oxidation, tryptophan oxidation, histidine oxidation, beta elimination, disulfide formation, disulfide scrambling, disulfide cleavage, and other changes that result in a change in the primary structure, including D-amino acid formation. Chemical instability, i.e. loss of chemical stability, can be examined by a variety of techniques, including ion exchange chromatography, capillary isoelectric focusing, analysis of peptide digests, and several types of mass spectrometry techniques. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical alterations may include size changes (e.g., clipping), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical alterations include charge changes (e.g., resulting from deamidation), which can be assessed by charge-based methods, such as, but not limited to, ion exchange chromatography, capillary isoelectric focusing, or peptide mapping.

[0112] Loss of physical and / or chemical stability may result in a change in biological activity, either as an increase or decrease in the biological activity of interest, depending on the modification and the protein modified. A protein "retains its biological activity" in a formulation if the biological activity of the protein at a given time is within about 30% of the biological activity exhibited at the time the formulation was prepared. Activity is considered to be reduced if the activity is less than 70% of its starting value. Biological assays may include both in vivo and in vitro based assays, such as ligand binding, potency, cell proliferation or other surrogate measures of its biopharmaceutical activity.

[0113] The term "naturally-occurring" as it occurs herein in reference to biological material, such as a polypeptide, a nucleic acid, a host cell, etc., refers to material that is found in nature.

[0114] The term "recombinant" indicates that a material (e.g., a nucleic acid or polypeptide) has been artificially or synthetically (i.e., non-naturally) changed by human intervention. Changes can be made to the material in its natural environment or state, or to material removed from its natural environment or state. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling, or other known molecular biology procedures. Examples of such molecular biology procedures can be found in Maniatis et al., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). A "recombinant DNA molecule," is comprised of segments of DNA joined together by such molecular biology techniques.

[0115] As used herein, the term "recombinant protein" or "recombinant polypeptide" refers to a protein molecule, e.g., a protein of interest, expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and / or expresses a recombinant nucleic acid.

[0116] The term "control sequence" or "control signal" refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is linked in a particular host cell. The nature of such control sequences may depend on the host organism. In certain aspects, prokaryotic control sequences may include a promoter, a ribosomal binding site, and a transcription termination sequence. Eukaryotic control sequences may include a promoter that includes one or more recognition sites for transcription factors, a transcription enhancer sequence or element, a polyadenylation site, and a transcription termination sequence. Control sequences may include leader sequences and / or fusion partner sequences. Promoters and enhancers consist of short arrays of DNA that interact specifically with cellular proteins involved in transcription (Maniatis, et al., Science 236:1237 (1987)). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect, and mammalian cells, as well as viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on which cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range, while others are functional in a limited subset of cell types (for reviews, see Voss, et al., Trends Biochem. Sci., 11:287 (1986) and Maniatis, et al., Science 236:1237 (1987)).

[0117] A "promoter" is a region of DNA that contains the site at which RNA polymerase binds and initiates transcription of messenger RNA by one or more downstream structural genes. Promoters are located near the transcription start site of a gene, on the same strand, and upstream of the DNA (towards the 5' region of the sense strand). Promoters are typically about 100-1000 bp in length.

[0118] An "enhancer" is a short (50-1500 bp) region of DNA that can bind to one or more activating proteins (transcription factors) to activate transcription of a gene.

[0119] As used herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the linking of nucleic acid sequences in such a way that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The term also refers to the linking of amino acid sequences in such a manner that a functional protein is produced. For example, a control sequence in a vector "operably linked" to a protein coding sequence is ligated thereto such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[0120] The term "polynucleotide" or "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers containing two or more nucleotide residues. The nucleotide residues that comprise a polynucleotide may be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates, and phosphoroamidates.

[0121] The term "oligonucleotide" refers to a polynucleotide containing 200 or fewer nucleotide residues. In some embodiments, the oligonucleotide is 10-60 bases in length. In other embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 nucleotides in length. The oligonucleotide may be single-stranded or double-stranded, for example, for use in constructing mutant genes. The oligonucleotide may be a sense oligonucleotide or an antisense oligonucleotide. The oligonucleotide may include a label, including a radioactive label, a fluorescent label, a hapten or an antigen label, for detection assays. The oligonucleotide may be used, for example, as a PCR primer, a cloning primer, or a hybridization probe.

[0122] A "polynucleotide sequence" or "nucleotide sequence" or "nucleic acid sequence", as used interchangeably herein, is a primary sequence of nucleotide residues in a polynucleotide, including oligonucleotides, DNA and RNA, nucleic acids, or character strings representing the primary sequence of nucleotide residues, depending on the context. From any particular polynucleotide sequence, either a given nucleic acid or a complementary polynucleotide sequence can be determined. It includes DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and represent the sense or antisense strand. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence discussed herein is the 5' end, and the leftward direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction, and the region of the sequence on the DNA strand that has the same sequence as the RNA transcript that is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence", and the region of the sequence on the DNA strand that has the same sequence as the RNA transcript that is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence".

[0123] As used herein, an "isolated nucleic acid molecule" or "isolated nucleic acid sequence" is a nucleic acid molecule that is either (1) identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid, or (2) cloned, amplified, tagged, or otherwise distinguished from background nucleic acids such that the sequence of the nucleic acid of interest can be determined. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that ordinarily expresses an immunoglobulin (e.g., an antibody), e.g., the nucleic acid molecule is in a different chromosomal location than in naturally occurring cells.

[0124] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of ribonucleotides along an mRNA strand, which also determines the order of amino acids along a polypeptide (protein) strand. Thus, the DNA sequence codes for the RNA sequence and the amino acid sequence.

[0125] The term "gene" is used broadly to refer to any nucleic acid associated with a biological function. A gene typically comprises a coding sequence and / or regulatory sequences required for expression of such a coding sequence. The term "gene" applies to a particular genomic or recombinant sequence, as well as to the cDNA or mRNA encoded by that sequence. Genes also include non-expressed nucleic acid segments that, for example, form recognition sequences for other proteins. Non-expressed regulatory sequences include transcriptional control elements to which regulatory proteins, such as transcription factors, bind and result in transcription of adjacent or nearby sequences.

[0126] "Expression of a gene" or "expression of a nucleic acid" means, as indicated by the context, transcription of DNA into RNA (optionally including modification of the RNA, such as splicing), translation of RNA into a polypeptide (possibly including subsequent post-translational modification of the polypeptide), or both transcription and translation.

[0127] Expression cassettes are a typical feature of recombinant expression technology. An expression cassette contains a gene encoding a protein of interest, e.g., a gene encoding an antibody sequence, such as an immunoglobulin light and / or heavy chain sequence. A eukaryotic "expression cassette" refers to a portion of an expression vector that allows for the production of a protein in a eukaryotic cell, such as a mammalian cell. It contains a promoter operable in a eukaryotic cell for mRNA transcription, one or more genes encoding the protein of interest, and mRNA termination and processing signals. An expression cassette can usefully contain, among the coding sequences, a gene useful as a selection marker. In the expression cassette, the promoter is operably linked 5' to the open reading frame encoding the exogenous protein of interest, and a polyadenylation site is operably linked 3' to the open reading frame. Other suitable control sequences can also be included, so long as the expression cassette remains operable. The open reading frame can optionally contain coding sequences for more than one protein of interest.

[0128] As used herein, the term "coding region" or "coding sequence" when used in reference to a structural gene refers to a nucleotide sequence that encodes the amino acids found in a nascent polypeptide as a result of translation of an mRNA molecule. In eukaryotes, the coding region is bounded on the 5' side by the nucleotide triplet "ATG", which encodes the initiator methionine, and on the 3' side by one of three triplets that specify a stop codon (i.e., TAA, TAG, TGA).

[0129] Recombinant expression techniques typically involve the use of a recombinant expression vector that includes an expression cassette and a mammalian host cell that includes the expression cassette or at least the recombinant expression vector carrying the expression cassette, for example, capable of being integrated into the host cell genome.

[0130] The term "vector" refers to any molecule or entity (eg, nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein coding information into a host cell.

[0131] The term "expression vector" or "expression construct" as used herein refers to a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of the operably linked coding sequence in a particular host cell. Expression vectors may include, but are not limited to, sequences that affect or control transcription, translation, and, if introns are present, RNA splicing of the coding region operably linked thereto. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence may also be encoded by the expression vector operably linked to the coding sequence of interest, if desired, so that the expressed polypeptide may be secreted by the recombinant host cell for easier isolation of the polypeptide from the cell. Such techniques are known in the art. (See, e.g., Goodey, Andrew R.; et al., Peptide and DNA sequences, U.S. Pat. No. 5,302,697; Weiner et al., Compositions and methods for protein secretion, U.S. Pat. No. 6,022,952 and U.S. Pat. No. 6,335,178; Uemura et al., Protein expression vector and utilization thereof, U.S. Pat. No. 7,029,909; Ruben et al., 27 human secreted proteins, U.S. Patent Application Publication No. 2003 / 0104400.) For expression of a multi-subunit protein of interest, an appropriate number and ratio of separate expression vectors, each containing the coding sequence for each of the different subunit monomers, can be used to transform the host cell. In other embodiments, a single expression vector can be used to express different subunits of a protein of interest.

[0132] The term "host cell" refers to a cell that has been transformed or can be transformed with a nucleic acid, thereby expressing a gene or coding sequence of interest. The term includes the progeny of a parent cell, whether or not the progeny is identical in morphology or genetic make-up to the original parent cell, so long as the gene of interest is present. Any of a large number of available and known host cells may be used in the practice of the invention to obtain antibody variants, although mammalian host cells capable of post-translationally glycosylating antibodies are preferred. The selection of a particular host depends on many factors recognized in the art. These include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, transformation rate, ease of collection of the peptide, expression characteristics, biological safety, and cost. These factors must be balanced with the understanding that not all hosts are equally effective at expressing a particular DNA sequence. Modifications can also be made at the DNA level. The DNA sequence encoding the peptide can be altered to codons more compatible with the selected host cell. Codons can be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. The transformed host is then cultured and purified. The host cells can be cultured under conventional fermentation conditions to express the desired compound. Such fermentation conditions are known in the art.

[0133] Within these general guidelines, microbial host cells in culture, such as bacteria (such as Escherichia coli species) and yeast cell lines (e.g., Saccharomyces, Pichia, Schizosaccharomyces, Kluyveromyces), as well as other fungal cells, algae or algae-like cells, insect cells, plant cells that have been engineered to incorporate humanized glycosylation pathways, can also be used to produce fully functional glycosylated antibodies. However, mammalian (including human) host cells, such as CHO cells and HEK-293 cells, are particularly useful in the processes of the invention.

[0134] Examples of useful mammalian host cell lines include CHO-K1 cells (e.g., ATCC CCL61), Chinese hamster ovary cells including CHO-S, DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture (Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster 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 CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocellular carcinoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al, Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells or FS4 cells; or mammalian myeloma cells, such as NS0 or sp2 / 0 mouse myeloma cells.

[0135] "Cells," "cell lines," and "cell cultures" are often used interchangeably, and all such designations herein include cell progeny. For example, a cell "derived from" a CHO cell is a cellular progeny of a Chinese Hamster Ovary cell, which may have been removed from the original primary cellular parent by any number of generations, and may also include transformed progeny cells. Transformants and transformed cells include the primary subject cell and cultures derived therefrom, regardless of the number of introductions. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0136] Host cells are transformed or transfected with the above-described nucleic acids or vectors for the production of polypeptides (including antigen binding proteins such as antibodies) and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Additionally, novel vectors and transfected cell lines having multiple copies of transcription units separated by a selectable marker are particularly useful for expressing polypeptides such as antibodies.

[0137] The term "transfection" refers to the uptake of foreign or exogenous DNA by a cell; a cell is "transfected" when exogenous DNA is introduced inside the cell membrane. Several transfection techniques are known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Using such techniques, one or more exogenous DNA moieties can be introduced into a suitable host cell.

[0138] The term "transformation" refers to a change in the genetic characteristics of a cell; a cell is transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed when it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques. After transfection or transduction, the transforming DNA may recombine with the cell's DNA by being physically integrated into the cell's chromosomes, or it may be maintained transiently as an episomal element without being replicated, or it may replicate independently as a plasmid. A cell is considered "stably transformed" if the transforming DNA is replicated with the division of the cell.

[0139] The process of the invention involves culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that allow the cells to secrete a protein of interest into the medium for a production culture period of at least 10 days.

[0140] Mammalian cells such as CHO and BHK cells are generally cultured as suspension cultures, i.e., the cells are suspended in liquid cell medium rather than attached to a solid support. Another useful production mode is a hollow fiber bioreactor with adherent cell lines. Porous microcarriers may be suitable and are commercially available under brands such as Cytoline®, Cytopore® or Cytodex® (GE Healthcare Biosciences).

[0141] "Cell culture" means extracellular medium (fresh or conditioned) and mammalian cells cultured therein.

[0142] "Cell culture medium" or "medium", as used interchangeably herein, is a sterile aqueous medium suitable for growing cells, preferably animal cells, more preferably mammalian cells (e.g., CHO cells) in in vitro cell culture. "Feed medium" is fresh cell culture medium added to a cell culture after cells have been inoculated into the cell culture medium and cell growth has commenced.

[0143] The term "production culture period" refers to the period during which protein-secreting mammalian cells are maintained under incubation conditions in a bioreactor that physiologically permits the continuous production of the protein of interest. In some embodiments, expression of the protein may be constitutive, while in other embodiments, expression of the protein can be engineered to be inducible (e.g., TetO-regulated expression). For such inducible expression, the production culture period includes only the period of culture in the bioreactor when an inducer molecule (e.g., tetracycline, doxycycline, or other tetracycline analogs) is present in the medium in an amount sufficient to induce expression of the protein of interest. For purposes of the claimed methods, the production culture period is at least 10 days or more, or at least 20 days or more, such as 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or more, or from 10 to 20 days or more, or from 20 to 30 days or more, or from 30 to 45 days or more, or from 45 to 60 days or more, or from 60 to 75 days or more.

[0144] During the production culture, fresh sterile liquid medium is automatically added to one or more perfusion bioreactors and mixed simultaneously from multiple different concentrated medium component solutions and aqueous diluents. The phrase "mixed simultaneously" means that the concentrated medium components and diluents are mixed together to create fresh culture medium, and mixed within seconds or minutes (≦2 min) as needed to replace the volume of medium removed from each of the perfusion bioreactors, either as permeate or cell bleed volume. Bioreactors have characteristic mixing times based on the bioreactor and impeller design, as well as the agitation speed. For example, the Xcellerex® XDR 500-L SUB has a blending time of 30-55 seconds at an agitation speed of 95-150 rpm. Shorter blending times are possible by increasing the agitation. The "permeate" is a volume of conditioned cell culture medium that has been filtered by microfiltration to remove all cells and contains the protein of interest. The conditioned medium upstream of the cell-removing microfilter is called the "retentate" and the conditioned medium downstream of the microfilter is the "permeate", which emerges from the perfusion system of the perfusion bioreactor and is ready for further processing, for example by a first chromatography system. The "cell bleed" is the volume of cell culture containing some cells and medium that is discharged from the bioreactor for disposal and / or analysis. Fresh medium is added to the bioreactor periodically or continuously, depending on whether the removal of the volume of cell culture from the bioreactor is performed intermittently (i.e., "periodically") or continuously.

[0145] In some embodiments of the process (and equipment) of the invention, fresh sterile liquid medium is added to one or more perfusion bioreactors by injecting multiple different concentrated component solutions in fixed ratios relative to each other directly into the perfusion bioreactor, while an aqueous diluent (suitable buffer or water) is also added in various ratios to the multiple different concentrated medium component solutions to maintain a constant culture volume in each perfusion bioreactor (i.e., to account for the volume of permeate or cell bleed that is being removed from each perfusion bioreactor). In other embodiments, fresh sterile liquid medium is added to one or more perfusion bioreactors by injecting multiple different concentrated component solutions and an aqueous diluent (suitable buffer or water) in fixed ratios relative to each other directly into the perfusion bioreactor to maintain a constant culture volume in each perfusion bioreactor. In yet other embodiments, fresh sterile liquid medium is added to one or more perfusion bioreactors by injecting multiple different concentrated component solutions and an aqueous diluent (suitable buffer or water) in fixed ratios relative to one another into a mixing chamber, where fresh sterile liquid medium is mixed simultaneously (in a sterile mixing vessel fluidly connected to the bioreactors) and then added to each perfusion bioreactor to maintain a constant culture volume.

[0146] The particular ratios in which the media components and diluents are suitably mixed will vary depending on the media recipe used and the concentrations of the concentrated media component stocks used, and appropriate ratios can be conveniently calculated by one of skill in the art.

[0147] According to the present invention, submerged addition of different concentrated media component solutions and aqueous diluents is preferably avoided. On-demand delivery of all media component solutions and aqueous diluents through separate ports can be achieved manually (e.g., by pre-set pumping flow rates with periodic adjustments, as needed) or automatically (e.g., by using ratio-controlled pumping skids and automation to maintain culture volume in the perfusion bioreactor).

[0148] The term "buffer" or "buffer solution" refers to a solution that resists changes in pH due to the action of its conjugate acid-base range. Examples of useful buffering agents include acetate, MES, citrate, tris, bis-tris, histidine, arginine, succinate, citrate, glutamate and lactate, or combinations of two or more thereof, or other mineral or organic acid buffers, with phosphate being another example of a useful buffering agent. Salts containing sodium, ammonium and potassium cations are often used to make buffered solutions.

[0149] A "domain" or "region" (used interchangeably herein) of a polynucleotide is any portion of an entire polynucleotide, up to and including, but typically less than, the complete polynucleotide. A domain can fold independently of the remainder of the polynucleotide chain (e.g., DNA hairpin folding) and / or can, but need not, correlate with a particular biological, biochemical, or structural function or location, such as a coding region or regulatory region.

[0150] A "domain" or "region" of a protein (used interchangeably herein) is any portion of the entire protein, up to and including, but typically less than, the complete protein. A domain can fold independently of the rest of the protein chain and / or can, but need not, correlate with a particular biological, biochemical, or structural function or location (e.g., a ligand-binding domain, or a cytoplasmic, transmembrane, or extracellular domain).

[0151] Quantitation of a protein of interest is often useful or necessary to track production and yield, or to properly formulate the protein or drug substance for further processing or storage. Thus, antibodies, particularly specific monoclonal antibodies, that specifically bind to a domain of the protein of interest can be useful for these purposes.

[0152] The term "antibody" or interchangeably "Ab" is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies (including human, humanized or chimeric antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, single chain antibodies, bispecific antibodies) capable of binding to antigen, comprising the complementarity determining regions (CDRs) of the foregoing, so long as they exhibit the desired biological activity. Multimers or aggregates of intact molecules and / or fragments are contemplated, including chemically derivatized antibodies. Antibodies of any isotype class or subclass are contemplated, including IgG, IgM, IgD, IgA, and IgE, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, or any allotype. Different isotypes have different effector functions, e.g., IgG1 and IgG3 isotypes have antibody-dependent cellular cytotoxicity (ADCC) activity.

[0153] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies, which are antigen-binding proteins, are highly specific binders to individual antigenic sites or epitopes, in contrast to polyclonal antibody preparations, which typically contain different antibodies to different epitopes. Non-limiting examples of monoclonal antibodies include mouse, rabbit, rat, chicken, chimeric, humanized, or human antibodies, fully assembled antibodies, multispecific antibodies (including bispecific antibodies), antibody fragments capable of binding to antigens (including Fab, Fab', F(ab)2, Fv, single chain antibodies, bispecific antibodies), maxibodies, nanobodies, and recombinant peptides comprising the aforementioned CDRs, or variants or derivatives thereof, so long as they exhibit the desired biological activity.

[0154] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, e.g., Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0155] The term "immunoglobulin" encompasses complete or partial antibodies, including two dimerized heavy chains (HC), each covalently linked to a light chain (LC), a single non-dimerized immunoglobulin heavy chain and a covalently linked light chain (HC+LC), or a chimeric immunoglobulin (light chain+heavy chain)-Fc heterotrimer (so-called "hemibody"), or a fusion protein, such as a peptibody, that contains a dimerized or non-dimerized Fc domain. An "immunoglobulin" is a protein, but not necessarily an antigen-binding protein, such as a carrier antibody covalently linked to a clinically relevant target binding moiety. On the other hand, immunoglobulins can be engineered to be bispecific or multispecific binders of multiple clinically relevant targets. The term "peptibody" refers to an antibody C. H 1. An antibody Fc domain (i.e., at least C) excluding CL, VH and VL domains and Fab and F(ab)2. H 2 and C H The term peptibody refers to a fusion protein molecule comprising an Fc domain (Fc domain, F ...

[0156] In an "antibody," each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain of about 220 amino acids (about 25 kDa) and one "heavy" chain of about 440 amino acids (about 50-70 kDa). The amino-terminal portion of each chain contains a "variable" ("V") region of about 100-110 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. The variable regions differ among different antibodies. The constant regions are the same among different antibodies. Within the variable region of each heavy or light chain, in the case of antibodies that are antigen-binding proteins, there are three hypervariable subregions that help determine the specificity of the antibody for the antigen. The variable domain residues between the hypervariable regions are called framework residues and are generally somewhat homologous among different antibodies. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domains of their heavy chains. Human light chains are classified as kappa (.kappa.) and lambda (.lambda.) light chains. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 or more amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). "Antibody" also encompasses recombinantly produced antibodies, and antibodies that are glycosylated or lack glycosylation.

[0157] The term "light chain" or "immunoglobulin light chain" includes full-length light chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length light chain comprises the variable region domain V L and constant region domain C L The light chain variable region domain is at the amino-terminus of the polypeptide. Light chains include kappa and lambda chains.

[0158] The term "heavy chain" or "immunoglobulin heavy chain" includes full-length heavy chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length heavy chain comprises the variable region domain V H, as well as three constant region domains C H1 , C H2 and C H3 Includes: V H The domain is located at the amino terminus of the polypeptide and is C H The domain is located at the carboxyl terminus and is C H3 is closest to the carboxy terminus of the polypeptide. Heavy chains are classified as mu (μ), delta (δ), gamma (γ), alpha (α) and epsilon (ε), which define the antibody's isotype as IgM, IgD, IgG, IgA and IgE, respectively. Heavy chains may be of any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE. Some of these may be further divided into subclasses or isotypes, e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Different IgG isotypes may have different effector functions (mediated by the Fc region), such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcγRs) on the surface of immune effector cells such as natural killers and macrophages, resulting in phagocytosis or lysis of the target cell. In CDC, antibodies kill target cells by triggering the complement cascade at the cell surface.

[0159] An "Fc region" or, as used interchangeably herein, an "Fc domain" or an "immunoglobulin Fc domain" refers to the C region of an antibody in an intact antibody. H1 and C H2 The two heavy chain fragments contain two or more disulfide bonds and a C domain. H3 The domains are held together by hydrophobic interactions.

[0160] The term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (eg, IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0161] For a detailed description of antibody structure and generation, see Roth, DB, and Craig, NL, Cell, 94:411-414 (1998), which is incorporated herein by reference in its entirety. Briefly, the process for generating DNA encoding heavy and light chain immunoglobulin sequences takes place primarily in developing B cells. Prior to rearrangement and joining of the various immunoglobulin gene segments, the V, D, J and constant (C) gene segments are generally found relatively close together on a single chromosome. During B cell differentiation, each one of the appropriate family members of the V, D, J (or only V and J in the case of light chain genes) gene segments recombine to form functionally rearranged variable regions of heavy and light immunoglobulin genes. This gene segment rearrangement process appears to be sequential. First, the heavy chain D-to-J joint is generated, followed by the heavy chain V-to-DJ joint and the light chain V-to-J joint. In addition to rearrangements of the V, D, and J segments, further diversity is generated in the primary repertoire of immunoglobulin heavy and light chains by variable recombination at the positions where the V and J segments of the light chain are joined, and where the D and J segments of the heavy chain are joined. Such light chain mutations typically occur within the last codon of the V gene segment and the first codon of the J segment. Similar joining imprecision occurs in the D and J segments. H It occurs on the heavy chromosome between the D and J segments and can span as many as 10 nucleotides. In addition, several nucleotides are between the D and J gene segments that are not encoded by genomic DNA. H Between gene segments and V H A gene segment may be inserted between the D gene segment and the D gene segment. These additions of nucleotides are known as N region diversity. The net effect of such rearrangements in the variable region gene segments and the variable recombination that may occur during such joining is the generation of a primary antibody repertoire.

[0162] The term "hypervariable" region refers to the amino acid residues of an antibody that are involved in antigen binding. The hypervariable region includes amino acid residues from the complementarity determining regions or CDRs (i.e., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, as described in Kabat et al., Sequences of Proteins of Immunological Interest, th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Even a single CDR can recognize and bind an antigen, but with a lower affinity than the entire antigen binding site including all the CDRs.

[0163] An alternative definition of residues from the hypervariable "loops" is described by Chothia et al., J. Mol. Biol. 196:901-917 (1987) as residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain.

[0164] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues.

[0165] The protein of interest may also be or include one or more antibody fragments. An "antibody fragment" comprises a portion of an intact full-length antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bispecific antibodies; linear antibodies (Zapata et al., Protein Eng., 8(10):1057-1062(1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0166] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment that contains the constant region. The Fab fragment contains all of the variable domains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fc fragment displays carbohydrates and is responsible for many of the antibody effector functions (such as binding complement and cellular receptors) that distinguish one class of antibody from another.

[0167] Pepsin treatment reduces the V H and V L F(ab')2 fragments are obtained that contain two "single-chain Fv" or "scFv" antibody fragments containing two domains, and these domains are present in a single polypeptide chain. Fab fragments differ from Fab' fragments by containing several additional residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Preferably, the Fv polypeptide further contains a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0168] A "Fab fragment" is a fragment of one light chain and one heavy chain. H1 and a variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0169] A "Fab' fragment" is a fragment that contains one light chain and one V H Domain and C H1 Domain and C H1 Domain and C H2 and a portion of one heavy chain, including the region between the two heavy chains, such that interchain disulfide bonds can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0170] "F(ab')2 fragment" is a fragment of two light chains and H1 Domain and C H2 The F(ab')2 fragment comprises two heavy chains containing a portion of the constant region between the heavy chains, resulting in the formation of an interchain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0171] "Fv" is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH VL dimer. A single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

[0172] A "single chain antibody" is an Fv molecule in which the heavy and light chain variable regions are linked by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single chain antibodies are described in WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference in their entireties.

[0173] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V H and V L the V domains are present in a single polypeptide chain and optionally contain a polypeptide linker that enables the Fv to form the desired structure for antigen binding. H Domain and V L The "Fd" fragment is located between the V H Domain and CH1 It consists of a domain.

[0174] The term "bispecific antibody" refers to a small antibody fragment that has two antigen-binding sites, which are bound to the same polypeptide chain (V H V L ) in the light chain variable domain (V L ) linked to a heavy chain variable domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are described more fully in, for example, EP 404097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0175] A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more V H The domains are covalently linked with a peptide linker to generate a bivalent domain antibody. H The regions may target the same or different antigens.

[0176] The term "antigen binding protein" (ABP) includes an antibody or antibody fragment, as defined herein, that specifically binds to a target ligand or antigen of interest.

[0177] In general, an antigen-binding protein, e.g., a protein of interest such as an immunoglobulin protein, or an antibody or antibody fragment, "specifically binds" to a target ligand or antigen of interest if it has a significantly higher binding affinity for that target ligand or antigen compared to its affinity for other unrelated proteins under similar binding assay conditions, such that the target ligand or antigen can be differentiated. Typically, an antigen-binding protein will have a dissociation constant (K D ) is 10 -8An antigen-binding protein is said to "specifically bind" its target antigen if its K D 10 -9 "high affinity" if K is less than or equal to M D 10 -10 If the molecule is M or less, it specifically binds to the antigen with "very high affinity."

[0178] "Antigen-binding region" or "antigen-binding site" refers to a portion of a protein that specifically binds to a particular target ligand or antigen. For example, that portion of an antigen-binding protein that contains amino acid residues that interact with a target ligand or antigen and confer to the antigen-binding protein its specificity and affinity for the antigen is called the "antigen-binding region". In an antibody, the antigen-binding region typically contains one or more "complementary binding regions" ("CDRs"). A particular antigen-binding region also contains one or more "framework" regions ("FRs"). A "CDR" is an amino acid sequence that contributes to antigen-binding specificity and affinity. The "framework" regions can help maintain the proper conformation of the CDRs to promote binding between the antigen-binding region and the antigen. In a conventional antibody, the CDRs are embedded within the framework of the heavy and light chain variable regions and constitute the regions involved in antigen binding and recognition. A variable region of an immunoglobulin antigen-binding protein comprises at least three heavy or light chain CDRs within framework regions (designated framework regions 1-4, FR1, FR2, FR3 and FR4 by Kabat et al., 1991; see also Chothia and Lesk, 1987), as referenced above (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1989, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883).

[0179] The term "target" or "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as an antigen binding protein (including, for example, an antibody, or an immunologically functional fragment of an antibody), and can be used in an animal to generate antibodies capable of binding to that antigen. An antigen can have one or more epitopes that can interact with different antigen binding proteins, such as antibodies.

[0180] The term "epitope" is the portion of a target molecule that is bound by an antigen-binding protein (e.g., an antibody or antibody fragment). The term includes any determinant capable of specific binding to an antigen-binding protein, such as an antibody or a T-cell receptor. An epitope may be contiguous or discontinuous (e.g., in a single chain polypeptide, amino acid residues that are not contiguous with one another in the polypeptide sequence but that are bound by the antigen-binding protein within the context of the molecule). In certain embodiments, an epitope may be a mimetic in that it comprises a three-dimensional structure similar to the epitope used to generate the antigen-binding protein, but does not contain, or only contains a portion of, the amino acid residues found in the epitope used to generate the antigen-binding protein. In most cases, epitopes are present on proteins, but in some instances, they may be present on other types of molecules, such as nucleic acids. Epitope determinants may include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. In general, an antigen binding protein specific for a particular target preferentially recognizes an epitope on that target in a complex mixture of proteins and / or macromolecules.

[0181] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" means the percent of identical residues between the amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) must be addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. For example, sequence identity can be determined by standard methods commonly used to compare the similarity of the amino acid positions of two polypeptides: using computer programs such as BLAST or FASTA, two polypeptide or two polynucleotide sequences are aligned for optimal matching of their respective residues (along the entire length of one or both sequences, or along a predetermined portion of one or both sequences).The program provides a default opening penalty and a default gap penalty, and a score matrix such as PAM 250 (a standard score matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)) can be used with the computer program. For example, the percent identity can then be calculated by multiplying the total number of identical matches by 100 and then dividing by the sum of the length of the longer sequence in the matched span and the number of gaps introduced into the longer sequence to align the two sequences. In calculating the percent identity, the sequences being compared are aligned to give the maximum match between the sequences.

[0182] The GCG program package is a computer program that can be used to determine percent identity, and this package includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or two polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acids or nucleotides (the "matched span" determined by the algorithm). A gap opening penalty (which is calculated as three times the average diagonal, where the "average diagonal" is the average of the diagonals of the comparison matrix being used, and the "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62, are used with the algorithm. In certain embodiments, standard comparison matrices (for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919) are also used by the algorithm.

[0183] Recommended parameters for determining percent identity of a polypeptide or nucleotide sequence using the GAP program include the following:

[0184] Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;

[0185] Comparison matrix: BLOSUM 62, 1992, by Henikoff et al., supra;

[0186] Gap penalty: 12 (but no penalty for end gaps)

[0187] Gap length penalty: 4

[0188] Similarity threshold: 0

[0189] A particular alignment scheme for aligning two amino acid sequences may result in matching only a short region of the two sequences, and this small aligned region may have very high sequence identity even if there is no significant relationship between the two full-length sequences. Therefore, the alignment method selected (GAP program) can be adjusted, if desired, to result in alignment over at least 50 consecutive amino acids of the target polypeptide.

[0190] The term "modification", when used in reference to a protein of interest, includes, but is not limited to, one or more amino acid changes (including substitutions, insertions or deletions); chemical modification; covalent modification by conjugation to a therapeutic or diagnostic agent; labeling (e.g., with radionuclides or various enzymes); covalent polymer attachment such as PEGylation (derivatization with polyethylene glycol) and the insertion or substitution of unnatural amino acids by chemical synthesis. By methods known to those of skill in the art, proteins can be "engineered" or modified for improved target affinity, selectivity, stability, and / or manufacturability before the coding sequence for the "engineered" protein is included in an expression cassette.

[0191] The terms "switch" or "switching," as used interchangeably herein with respect to a protein isolate fraction, a purified product pool, a virus-free filtrate, or another pool, fraction, eluate, or resultant liquid effluent from a process step or equipment component, means to fluidly direct, divert, direct, channel, or convey that effluent to a subsequent process step or equipment component. Such switching can be under automated control and regulation of computers and / or robotic mechanisms (e.g., valves or pumps), or can be manually controlled and regulated.

[0192] The term "switchable" in reference to a collection vessel, surge vessel, holding vessel, mixing vessel, tank, bag, conduit, pipe, tube, or other conveying means that liquid can flow into or out of the vessel, or that such flow can be switched, directed, diverted, directed, diverted, or fluidly conveyed to a different vessel, tank, conduit, pipe, tube, or conveying means. Such switching can be under automatic control and regulation of a computer and / or robotic mechanism (e.g., valves or pumps), or can be manually controlled and regulated.

[0193] The term "surge receptacle" refers to a storage reservoir, mixing vessel, feed tank, or collection vessel (or interchangeably "collection tank") at the downstream end of a conduit, feeder, dam, pipe, or tube to absorb mismatched flow rates between two fluidly connected unit operations, e.g., the flow rate of permeate coming from a bioreactor and the flow rate of a first chromatography system under automated control in a continuous or semi-continuous format process embodiment of the present invention. The surge receptacle absorbs the change or difference in flow rates by surging a volume within pre-set volume range limits between the fluidly connected unit operations (see, e.g., FIG. 3). For purposes of the present invention, a surge receptacle typically includes a maximum volume of 50-650 L, with 100-650 L vessels being most useful in semi-continuous process embodiments, while 50-200 L vessels are usually sufficient in continuous process embodiments. In some embodiments of the invention, operations downstream of the viral inactivation system / neutralization system involve batch processing of a viral inactivation product pool, which may optionally be filtered by depth filtration to obtain a filtered viral inactivation product pool (FVIP), which in such embodiments may be collected in a collection vessel, and in subsequent batch steps or operations, the purified product pool or virus-free filtrate may be collected in other collection vessels between steps. In such separate operations, batch or batch mode processing, the collection vessel or interchangeably "collection tank" (which in certain embodiments may also be considered a "feed tank" for subsequent steps) from one step lacks the automated control of a surge vessel, and although the collection vessel (or feed tank) may be physically similar to a surge vessel, for purposes of the present invention, such collection vessel (or interchangeably "collection tank") or feed tank is referred to as a "holding vessel" or interchangeably "HV" (e.g., HV1, HV2, HV3, HV4, or HV5). A "holding vessel" may be a disposable holding vessel (SUHV) that is distinct from a disposable collection vessel (SUCV, e.g., SUCV1 or SUCV2) in a continuous or semi-continuous format set of manufacturing process steps or operations.

[0194] A "chromatographic system" is an arrangement of at least one enclosed chromatographic matrix with closed conduit hardware (e.g., pipes or tubes) for fluid ingress and egress from at least one chromatographic matrix. The chromatographic system includes one or more pumps and / or valves for automatic or manual control of fluid flow rate and pressure. The first, second and third chromatographic systems of the process and equipment of the present invention can incorporate various types of chromatographic matrices, and those skilled in the art will know how to select and use in sequence as appropriate for the protein of interest. For purposes of the method of the present invention, the term "matrix" encompasses resins, beads, nanoparticles, nanofibers, hydrogels, membranes (e.g., membrane adsorbers (MA)), and monoliths, or any other physical matrix, with associated covalently attached chromatographic ligands (e.g., Protein A, Protein G, or other affinity chromatographic ligands, such as targeting ligands, charged moieties, or hydrophobic moieties). The matrix to which the affinity targeting ligand is attached is most often agarose, but other matrices are available. For example, mechanically stable matrices such as controlled pore glass, methacrylate (e.g., Amsphere™ A3 resin, JRS Life Sciences) or poly(styrenedivinyl)benzene allow for higher stability, faster flow rates and shorter processing times than can be achieved with agarose. If the protein contains a CH3 immunoglobulin domain, Bakerbond ABX™ (JT Baker, Phillipsburg, NJ) resin can be useful for purification. The affinity chromatography matrix can be arranged or packed into a column that is useful for purifying the protein. For example, the loading of the cell-free culture fraction onto the affinity chromatography matrix in the first chromatography system is preferably performed at about neutral pH.

[0195] The term "binding" or "binding" of a molecule to Protein A or a Protein A matrix or another (distinct) affinity chromatography matrix means exposing the molecule to an affinity chromatography ligand covalently attached to a solid substrate (e.g., a resin) under appropriate conditions (e.g., pH and selected salt / buffer composition) such that the molecule of interest becomes reversibly immobilized in or on the affinity chromatography ligand by virtue of its binding affinity under those conditions, regardless of the physical mechanism of affinity that may be involved. (See, e.g., Jendeberg, L. et al., The Mechanism of Binding Staphylococcal Protein A to Immunoglobulin G Does Not Involve Helix Unwinding, Biochemistry 35(1):22-31(1996); Nelson, JT et al., Mechanism of Immobilized Protein A Binding to Immunoglobulin G on Nanosensor Array Surfaces, Anal. Chem., 87(16):8186-8193(2015)).

[0196] The term "binding" or "binding" of a molecule to an ion exchange matrix (e.g., a CEX matrix such as a CEX resin or membrane adsorber, or an AEX matrix such as an AEX resin or membrane adsorber) means exposing the molecule to the ion exchange matrix under appropriate conditions (e.g., pH and selected salt / buffer composition) such that the molecule becomes reversibly immobilized in or on the ion exchange matrix by ionic interactions between the molecule and the charged group(s) (i.e., charged ligands) of the ion exchange matrix.

[0197] The term "loading buffer" or "equilibration buffer" refers to the buffer and salts that are mixed with a protein preparation (e.g., the permeate or filtrate of a batch or perfusion cell culture, or the eluate pool containing the protein of interest) to load the protein preparation onto a Protein A matrix or other affinity chromatography matrix, or onto an ion exchange matrix (e.g., a CEX matrix or an AEX matrix), or onto a hydrophobic interaction chromatography (HIC) matrix, as the case may be. This buffer is also used to equilibrate the chromatography matrix before loading and to wash after loading the protein.

[0198] The term "wash buffer" is used herein to refer to a buffer that is passed through a Protein A matrix or another affinity chromatography matrix, or an ion exchange matrix (e.g., a CEX matrix or an AEX matrix), or a hydrophobic interaction chromatography (HIC) matrix, as the case may be, after loading of a protein preparation and before elution or flow-through of the protein of interest. The wash buffer may serve to remove one or more contaminants without substantial elution of the desired protein, or may be used to wash away unbound proteins.

[0199] The term "elution buffer" or "elution solution" refers to a buffer used to elute a protein of interest that is reversibly bound to a matrix. As used herein, the term "solution" refers to either a buffered solution, including water, or a non-buffered solution.

[0200] The term "elution pool" or "eluate pool" refers to the material that has been eluted from the matrix, which material contains the recombinant protein of interest.

[0201] The term "loading", with respect to a Protein A matrix or other affinity chromatography matrix, or ion exchange matrix (e.g., a CEX matrix), or hydrophobic interaction chromatography (HIC) matrix, means filling the Protein A matrix or another affinity chromatography matrix, or ion exchange matrix, or HIC matrix with a protein preparation (e.g., the permeate or filtrate of a batch or perfusion cell culture, or an eluate pool containing the protein of interest).

[0202] The term "washing" with respect to a Protein A matrix or other affinity chromatography matrix, or an ion exchange matrix (e.g., a CEX matrix or an AEX matrix), or an HIC matrix, as the case may be, means passing an appropriate buffer through the Protein A matrix or the ion exchange matrix or the HIC matrix or other chromatography matrix.

[0203] The term "elution" of a molecule (e.g., a desired recombinant protein or a contaminant) from a Protein A matrix or another affinity chromatography matrix, or an ion exchange matrix (e.g., a CEX matrix or an AEX matrix), or a HIC matrix, refers to the removal of the molecule from such material, typically by passing an elution buffer over the chromatography matrix.

[0204] The terms "single-use" or "single use" components, used interchangeably, mean that a particular sterile production line component, i.e., sterile equipment, used in the automated facility of the invention or in carrying out the process of the invention is constructed or configured to be used for a single production run (but may be reused if quality and sterile hygiene can be guaranteed for multiple runs). The disposable component can then be disposed of and replaced for a subsequent production run by another disposable component of the same or modified configuration without the need for cleaning and disinfecting the component between production runs. Examples of disposable components that can be used in the present invention include, but are not limited to, a perfusion bioreactor, a first chromatography system, a second chromatography system, a third chromatography system, a low pH or detergent viral inactivation system, a neutralization system, a viral filtration system, or an ultrafiltration / diafiltration system. Such disposable components can be constructed or obtained commercially, for example, but are not limited to:

[0205] Disposable bioreactors: XCelleex® XDR Disposable Bioreactor Bags (e.g., 500L, 1000L, or 2000L volumes; GE Healthcare Life Sciences); BIOSTAT STR® Stirred Tank Disposable Bioreactor Systems (e.g., 500L-2000L volumes; Sartorius Stedim Biotech); HyPerforma Disposable Bioreactors (e.g., 50L, 100L, 200L, 500L, 1000L, and 2000L volumes; Thermo Fisher Scientific); Allegro™ Disposable Stirred Tank Bioreactors (e.g., 500L-2000L volumes; Pall); Millipore Mobius® Disposable Bioreactors (e.g., 500L-2000L volumes; MilliporeSigma), including but not limited to Wave Bioreactor® Bags (GE Healthcare Life Sciences) or RIM Bio Rocker 50 L rocking bioreactor bags including Bags; or mixer bags available from Pall or Sartorius (e.g., 100 L, 200 L, 650 L, 1000 L or 2000 L volumes);

[0206] Disposable perfusion systems: Spectrum Krosflo® hollow fiber system or Repligen alternating tangential flow (ATF-6 and 10) systems;

[0207] Disposable Heat Exchanger: Thermo Scientific™ DHX™ Heat Exchanger with Thermo Scientific™ ThermoFlex™ Recirculating Chiller, and Thermo Scientific™ DHX™ Bag Assembly;

[0208] Disposable filter assembly systems including filters (various membranes and pore sizes from MilliporeSigma or Sartorius Stedim Biotech), silicone and / or c-flex tubing, and sterile connectors (Pall, Colder, GE Healthcare Life Sciences, Sartorius Stedim Biotech);

[0209] Disposable transfer lines in various sizes, lengths and configurations using disposable sterile connectors, silicone and / or c-flex type tubing are commercially available from Thermo Fisher Scientific (ASI) or Advantapure;

[0210] Disposable media component solution or aqueous diluent (e.g., buffer) solution tote storage bags are commercially available from Advanced Scientific (ASI; Thermo Fisher Scientific), MilliporeSigma, Sartorius, or RIM Bio;

[0211] Disposable viral inactivation systems: Cadence® viral inactivation system manifold (Pall Life Sciences), FlexAct® for low pH viral inactivation ("VI"; Sartorius); disposable chromatography systems: Cadence™ BioSMB® PD (Pall Life Sciences); Allegro™ disposable chromatography (Pall Biotech); Mobius® FlexReady chromatography (MilliporeSigma); AKTA™ Ready disposable chromatography (GE Healthcare Life Sciences); or Sartobind® IEX membrane adsorbers (Sartorius Stedim Biotech);

[0212] Disposable viral filtration systems: Allegro™ MVP Disposable System Manifold (Pall Biotech); Mobius® FlexReady for viral filtration (Millipore Sigma); FlexAct® (Sartorius), Planova™ Disposable Virus Filtration (SU-VFS; Asahi Kasei Bioprocess America), or Viresolve® Pro Virus Filtration (Millipore Sigma);

[0213] Disposable UF / DF systems: Allegro™ Disposable Tangential Flow Filtration System (Pall Biotech); Mobius® FlexReady TFF System (MilliporeSigma); FlexAct® for UF / DF (Sartorius); AKTA™ Readyflux Disposable Filtration (GE Healthcare Life Sciences); and

[0214] Disposable sterile connectors: AseptiQuik® connectors (Colder Products Company), Kleenpak® Presto sterile connectors (Pall Biotech); Lynx® ST connectors (MilliporeSigma).

[0215] The terms "filter bank" or "filter assembly system", used interchangeably, refer to an apparatus that includes a plurality of filter assemblies, each filter assembly including at least one filter. The filters included in the filter assemblies are disposable filters and can be replaced after a period of time and / or amount of use. The filter bank can be a portable device. For example, the filter bank can be placed on a filtration cart that can be moved to various locations in an automated facility. The filters included in the filter bank can include a filtration system including a depth filter, a 0.2 micrometer filter, a membrane filter, a 20 nanometer (nm) filter, a virus filtration device, an ultrafiltration device, a diafiltration device, or a combination thereof. The filter bank can be configured such that material flows through at least one filter of the filter bank while another filter of the filter bank remains unused. In various embodiments, the filter bank can be coupled to a diverter valve or other flow control device to control the flow of material to the filters included in the filter bank. The diverter valve or flow control device can be pneumatically controlled.

[0216] The above is merely an illustrative, and not an exhaustive, list of disposable systems and connectors available to one of ordinary skill in the art of the present invention.

[0217] Protein of interest

[0218] The protein of interest produced using the present invention may be any industrially or medically useful protein, such as, but not limited to, a pharmacologically active protein or peptide.

[0219] For example, the protein of interest may be a peptide mimetic or an agonist peptide. The terms "peptide mimetic," "peptide mimetic," and "agonist peptide" refer to a peptide or protein that has a biological activity comparable to a naturally occurring protein of interest. These terms include peptides that indirectly mimic the activity of a naturally occurring peptide molecule, such as by enhancing the effect of the naturally occurring molecule.

[0220] The protein of interest may be an antagonist peptide or an inhibitor peptide. The terms "antagonist peptide," "peptide antagonist," and "inhibitor peptide" refer to a peptide or protein that blocks or in some way interferes with the biological activity of a receptor of interest, or that has biological activity comparable to a known antagonist or inhibitor of the receptor of interest (e.g., but not limited to, an ion channel or a G protein-coupled receptor (GPCR)).

[0221] Examples of pharmacologically active proteins that can be produced according to the present invention include IL-6 binding peptides, CD3 binding proteins, CD19 binding proteins, CD20 binding proteins, CD22 binding proteins, HER2 binding proteins, HER3 binding proteins, vascular endothelial growth factor-A (VEGF-A) binding proteins, TNF-α binding proteins, EGFR binding proteins, RANK ligand binding proteins, IL-1α binding proteins, IL-1β binding proteins, IL-17A binding proteins, EPCAM (CD326) binding proteins, CGRP peptide antagonists, bradykinin B1 receptor peptide antagonists, toxins, These include, but are not limited to, peptides, placental growth factor (PIGF) binding proteins, parathyroid hormone (PTH) agonist peptides, parathyroid hormone (PTH) antagonist peptides, ang-1 binding peptides, ang-2 binding peptides, myostatin binding peptides, erythropoietin mimetic (EPO mimetic) peptides, FGF21 peptides, thrombopoietin mimetic (TPO mimetic) peptides (e.g., AMP2 or AMPS), nerve growth factor (NGF) binding peptides, B cell activating factor (BAFF) binding peptides, and glucagon-like peptide (GLP)-1 or a peptidomimetic thereof or GLP-2 or a peptidomimetic thereof.

[0222] Proteins and coding sequences for such proteins, some of which have already received regulatory approval, are known in the art, but the invention may also be applied to the manufacture of drug substances that have not yet been revolutionized by the methods of drug discovery, research and development, and clinical trials.

[0223] DNA cloning

[0224] Cloning of DNA is performed using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, incorporated herein by reference). For example, a cDNA library can be constructed by reverse transcription of polyA+ mRNA, preferably membrane-bound mRNA, and the library screened with a probe specific for human immunoglobulin polypeptide gene sequences. However, in one embodiment, polymerase chain reaction (PCR) is used to amplify the cDNA (or a portion of a full-length cDNA) encoding the immunoglobulin gene segment of interest (e.g., a light chain variable segment or a heavy chain variable segment). The amplified sequence can be readily cloned into any suitable vector, e.g., an expression vector, a minigene vector, or a phage display vector. It will be appreciated that the particular cloning method used is not critical, so long as it is possible to determine the sequence of some portion of the protein of interest.

[0225] One source of antibody nucleic acid is a hybridoma produced by obtaining B cells from an animal immunized with an antigen of interest and fusing them with immortal cells. Alternatively, the nucleic acid can be isolated from the B cells (or the whole spleen) of the immunized animal. Yet another source of nucleic acid encoding an antibody is a library of such nucleic acids, for example, produced by phage display technology. Polynucleotides encoding peptides of interest, for example variable region peptides with desired binding properties, can be identified by standard techniques such as panning.

[0226] DNA sequencing is performed using standard techniques (e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467, which are incorporated herein by reference). By comparing the sequence of the cloned nucleic acid with published sequences of genes and cDNAs, one of skill in the art could easily determine which regions have been sequenced. One source of gene sequence information is the National Center for Biotechnology Information at the National Library of Medicine, National Institutes of Health, Bethesda, Maryland. Gene sequencing can also be performed, for example, by standard methods or by so-called "next generation" sequencing of engineered DNA constructs prior to transfection. (See, e.g., Buermans, HPJ, & den Dunnen, JT, Next generation sequencing technology: Advances and applications, Biochimica et Biophysica Acta-Molecular Basis of Disease 1842(10):1932-1941 (2014)).

[0227] Chemical synthesis of part or all of the coding region containing the codons reflecting the desired protein change can be cloned into an expression vector by either restriction digestion and ligation of the 5' and 3' ends of the entire open reading frame (ORF) or fragments containing nucleotide overhangs that are compatible with the destination vector, generated by restriction enzyme digestion. The fragment or insert is typically ligated into the destination vector using T4 ligase or other common enzymes. Other useful methods are similar to the above, except that the restriction enzyme cleavage site is at a different position than the recognition sequence. Alternatively, isothermal assembly (i.e., "Gibson Assembly") can be used, where nucleotide overhangs are generated during synthesis of the fragment or ORF, and digestion with an exonuclease is used. Alternatively, the nucleotide overhangs can be ligated ex vivo by ligase or polymerase, or in vivo by intracellular processes.

[0228] Alternatively, homologous recombination can be used, similar to isothermal assembly, except that the exonuclease activity of T4 DNA ligase can be used on both the insert and the vector, and ligation can be performed in vivo.

[0229] Another useful cloning method is the so-called "TOPO" method, in which a complete insert containing a 3' adenosine overhang (generated by Taq polymerase) is presented and topoisomerase I ligates the insert into a TOPO vector.

[0230] Another useful cloning method is degenerate or error-prone PCR, which utilizes degenerate primers and / or a thermostable low-fidelity polymerase triggered by the polymerase within certain reaction conditions. The fragment or insert is then cloned into an expression vector.

[0231] The above are only examples of known cloning techniques, and one of skill in the art will know how to use any other suitable cloning technique.

[0232] The isolated DNA can be operably linked to a control sequence or placed into an expression vector, which is then transfected into a host cell that does not produce immunoglobulin proteins to direct the synthesis of monoclonal antibodies in the recombinant host cell. Recombinant production of antibodies is known in the art.

[0233] A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0234] Many vectors are known in the art. The vector components may include one or more of the following: a signal sequence (which may, for example, direct secretion of an expressed protein by a recombinant host cell), an origin of replication, one or more selectable marker genes (which may, for example, confer antibiotic or other drug resistance, complement an auxotrophic deficiency, or supply a critical nutrient not available in the medium), an enhancer element, a promoter, and a transcription termination sequence, all of which are known in the art.

[0235] Protein expression

[0236] The method of the invention for producing a purified protein of interest (such as, but not limited to, a protein drug substance) includes culturing protein-secreting mammalian cells. Such cultured mammalian cells are typically generated by recombinant DNA techniques including transient or stable transfection, e.g., pooled plasmid constructs (expression vectors) from the cloning step can be transfected into multiple host cells (e.g., mammalian, e.g., HEK293 or CHO, bacterial, insect, yeast cells) for expression using cationic lipids, polyethylenimine, Lipofectamine™ or ExpiFectamine™, or electroporation. Those skilled in the art will recognize numerous suitable means for transfecting to achieve expression of recombinant antibodies. Alternatively, production cell lines of protein-secreting mammalian cells can be created using methods for stable genomic integration of an expression cassette encoding a protein of interest.(See, e.g., Zhang, Crispr-Cas Systems and Methods for Altering Expression Of Gene Products, WO 2014093661; Frendewey et al., Methods and Compositions for Targeted Modification of a Genome, U.S. Patent No. 9,228,208; Church et al., Multiplex Automated Genome Engineering, WO 2008052101, U.S. Patent No. 8,153,432; Bradley et al., Methods Cells and Organisms, U.S. Patent Application Publication No. 2015 / 0079680; Begemann et al., Compositions and Methods for Modifying Genomes, WO 2017141173; Gill et al., Nucleic acid-guided nucleases, U.S. Patent No. 9,982,279; Minshull et al., Enhanced nucleic acid constructs for eukaryotic genes (See, e.g., U.S. Patent Nos. 9,428,767, 9,580,697, 9,574,209; Minshull et al., DNA Vectors, Transposons And Transposases For Eukaryotic Genome Modification, U.S. Patent No. 10,041,077).

[0237] Optionally, the transfectant or transformant cell is provided with a recombinant expression cassette for one or more of the proteins that bind to a selectable marker, such as, but not limited to, glutamine synthase, dihydrofolate reductase, puromycin-N-acetyltransferase, blasticidin-S deaminase, hygromycin phosphotransferase, aminoglycoside phosphotransferase, nourseothircin N-acetyltransferase, or zeocin.

[0238] The protein of interest is typically obtained by culturing transfected or transformed host cells under physiological conditions that allow the cells to express the recombinant protein. Most conveniently, the expressed recombinant protein is directly secreted into the extracellular medium (by using an appropriate secretion-directing signal peptide) and collected therefrom, otherwise further steps are required to isolate the expressed antibody from a cell extract.

[0239] The desired scale of recombinant expression depends on the type of expression system and the desired amount of protein product. Some expression systems, such as ExpiCHO™, usually produce higher yields compared to some earlier HEK293 technologies. Therefore, a smaller scale of ExpiCHO™ may be sufficient compared to the HEK293 system. The efficiency of transfection may also be a consideration when selecting an appropriate expression system. Electroporation may be a suitable method given its effectiveness, relatively low cost, and the fact that high throughput during this step is not critical. Furthermore, the ratio of immunoglobulin light to heavy chains can be varied during co-transfection to improve expression of a particular variant. The product yield of a given variant must be sufficient to withstand multiple handling steps and generate a signal high enough to be detected by the chosen fluorescence detector.

[0240] In general, transfected or transformed host cells are typically cultured by any conventional type of culture, such as batch, fed-batch, enhanced fed-batch, or continuous. Suitable continuous cultures included repeated batch, chemostat, turbidostat, or perfusion culture with product and cell retention or cell retention only. However, for purposes of the present invention, the culture is carried out in one or more disposable perfusion bioreactors, each of which can contain a volume of liquid medium from about 50 L to about 4000 L (e.g., 50 L, 60 L, 75 L, 100 L, 250 L, 500 L, 650 L, 750 L, 1000 L, 1250 L, 1500 L, 1750 L, 2000 L, 2250 L, 2500 L, 2750 L, 3000 L, 3250 L, 3500 L, 3750 L, or 4000 L) as desired. The number of disposable bioreactors used to culture the cells is 1, 2, 3, 4, 5, or 6 disposable perfusion bioreactors of the desired volume.

[0241] The host cells used in the present invention to produce a protein of interest or "POI" (e.g., a non-glycosylated or glycosylated protein) can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. Additionally, see Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., J. Clin. Microbiol. 1999, 11:1311-1323 (1989); and W. et al., J. Clin. Microbiol. 1999, 11:1311-1323 (1999). Any of the media described in U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90103430; WO 87 / 00195; or U.S. Pat. No. 30985 may be used as a culture medium for the host cells. Any of these media may contain hormones and other nutrients such that the physiological conditions of the cells in or on the medium promote expression of the protein of interest by the host cells. and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, phosphate, etc.), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug Gentamicin™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range) and glucose or an equivalent energy source can be supplemented as necessary, and any other necessary supplements can also be included at appropriate concentrations that would be known to those of skill in the art.

[0242] The predetermined culture conditions, such as temperature (typically, but not necessarily, about 37±1° C. for mammalian cells), pH (typically, but not necessarily, cell culture media are maintained within a range of about pH 6.5-7.5), oxygenation, etc., will be apparent to one of skill in the art. By "culturing" or "maintaining" at a predetermined culture condition, it is meant that the process control system is set to a particular value for that condition, in other words, the intended volume, target temperature, pH, oxygenation level, etc., that is maintained at a predetermined set point for each parameter within a narrow range (i.e., a "narrow deadband") that is most suitable for the cell line and protein product of interest. Obviously, there will be small variations in temperature, pH, or other culture conditions over time and from location to location throughout the culture vessel (i.e., bioreactor). (For example, Oguchi et al., pH Condition in temperature shift cultion enhances cell longevity and specific hMab productivity in CHO culture,Cytotechnology.52(3):199-207(2006);Al-Fageeh et al.,The cold-shock response in cultured mammalian cells:Harnessing the response for improvement of recombinant protein production,Biotechnol.Bioeng.93:829-835(2006); see also Marchant, RJet al., Metabolic rates, growth phase, and mRNA levels influencing cell-specific antibody production levels from in vitro cultured mammalian cells at sub-physical temperature,Mol.Biotechnol.39:69-77(2008)).

[0243] Digital control units and sensor monitors are commercially available or can be constructed by those skilled in the art. Alternative digital control units (DCUs) that control and monitor cell culture processes are manufactured and commercially available by companies such as B.Braun, New Brunswick, Sartorius, or Thermo Fisher Scientific. Table 1A (below) lists some examples of digital control and sensor devices that can be used to monitor cell culture conditions. Other online or offline analyses can include off-gas measurements by mass spectrometry, detailed determination of medium composition (amino acids, vitamins, trace minerals), and extensive testing of cellular metabolic products other than CO2 and lactate.

[0244] [Table 1A]

[0245] The medium may contain an appropriate amount of serum, such as fetal bovine serum (FBS), or preferably, the host cells may be adapted for culture in serum-free medium. In some embodiments, the aqueous medium is liquid, such that the host cells are cultured in a cell suspension within the liquid medium. The host cells may be usefully grown in continuous (perfusion) cell culture systems, preferably designed for disposable use.

[0246] According to the present invention, fresh media is mixed simultaneously from multiple concentrated component solutions and an aqueous diluent. Cell media are complex mixtures that contain a wide range of concentrations of each component as well as unique ratios of one component to another. The factor by which any cell media formulation can be concentrated is limited by the solubility, stability, or filterability of its least soluble, least stable, or least filterable component. By dissolving components as chemically compatible subgroups, an increase in concentration factor can be achieved that would not be possible if all components were dissolved together. For example, some components are more soluble at acidic pH and others are more soluble at alkaline pH. In this example, components soluble at acidic pH can be grouped together in one solution while components soluble at alkaline pH can be grouped together in another solution that, when recombined, form a complete media. In addition to or instead of pH grouping to achieve higher concentrations, other solvents such as alcohol or dimethyl sulfoxide (DMSO) can be utilized, or stock solutions can be created for individual components that have special solubility or storage requirements that require exclusion from other components until added to the bioreactor. The exact grouping of compatible components and their maximum concentrations for any given cell media formulation are readily determined by one of ordinary skill in the art.

[0247] Typically, the viable cell density is about 1.0 x 10 6 ~about 2×10 8 Cells / mL, e.g. 1.0 x 10 6 ~2.0×10 7 in the range of cells / mL, or approximately 4 x 10 7 cells / mL ~ approx. 5×10 7 In the range of cells / mL, or approximately 1 x 10 8 cells / mL ~ approx. 2×10 8 A range of 1000 to 2000 cells / mL can be used. It is known that increasing the concentration of cells toward the upper end of the preferred range can improve volumetric productivity. Nevertheless, a range of cell densities is envisioned that includes any of the above point values ​​as the lower or upper end of the range. The desired scale of recombinant expression and cell culture depends on the type of expression system and the amount of drug substance desired.

[0248] For the purposes of the claimed invention, the transfected or transformed host cells are cultured such that the recombinant polypeptide or protein is secreted directly into the medium. Harvesting the recombinant protein involves separating it from the particulate matter, which may include host cells, cell aggregates, and / or lysed cell fragments, into a cell-free fraction free of host cells and cell debris, i.e., a cell-free "permeate". Such cells and cell debris are removed from the conditioned medium, for example, by centrifugation and / or microfiltration. For example, to make the permeate, a series of filtration steps, such as hollow fiber membranes (0.2 μm pore size) or depth filtration, can be used, which can be mobile, replaceable, and / or disposable, and "filtration carts".

[0249] Some embodiments of the invention include a first disposable surge vessel (SUSV1) adapted to receive a volume of permeate removed from a perfusion bioreactor, the permeate volume being cell-free. These permeate volumes are automatically and fluidly delivered to SUV1 from one or more disposable perfusion bioreactors. In some embodiments, there is an automatic controller including a detector for measuring the fluid volume of SUSV1 and a processor for varying the pump speed of the first chromatography system to maintain a preset volume range of SUSV1.

[0250] In some embodiments of the present invention, the equipment for carrying out the process further includes a hollow fiber membrane, a series of depth filters, or a filtration cart to render the permeate cell-free before it is automatically and fluidly fed to SUSV1.

[0251] Protein purification and viral inactivation

[0252] In general, purification of proteins (e.g., recombinant or naturally occurring proteins) is usually accomplished by any series of chromatographic steps such as anion exchange chromatography, cation exchange chromatography, affinity chromatography (using Protein A or Protein G or Protein L as the affinity ligand or another distinct affinity ligand), hydrophobic interaction chromatography (HIC), hydroxyapatite chromatography, reverse phase HPLC, and size exclusion chromatography. The above are non-limiting examples of chromatographic modalities that can be included in either the first chromatographic system, the second chromatographic system, and / or the third chromatographic system. Each of the first, second or third chromatography systems can be configured with one, two, three or more different chromatography matrices (e.g., chromatography columns) fluidly linked, preferably in series, as required for the protein of interest, and can optionally be arranged on a mobile, replaceable or disposable disposable unit, skid or "cart." Additionally, the purification process can include one or more ultrafiltration, nanofiltration or diafiltration steps.

[0253] Any other known techniques for protein purification, such as ethanol precipitation, isoelectric focusing, SDS-PAGE and ammonium sulfate precipitation, are also possible depending on the protein collected.

[0254] In the process of the present invention for producing a purified protein of interest (e.g., a protein drug substance), the protein of interest (e.g., but not limited to, a protein drug) in the cell-free permeate is captured by one or more chromatographic capture steps in a first chromatographic system that can partially purify and / or concentrate the protein, such as, but not limited to, Protein A or Protein G or Protein L affinity chromatography, or affinity chromatography using different affinity ligands covalently bound to a solid matrix. (See, e.g., Frank, MB, "Antibody Binding to Protein A and Protein G beads" 5. In: Frank, MB, ed., Molecular Biology Protocols. Oklahoma City (1997)). The first chromatography system can optionally include anion exchange chromatography (AEX), cation exchange chromatography (CEX), affinity chromatography (using Protein A or Protein G or Protein L as an affinity ligand or another specific targeting moiety), hydrophobic interaction chromatography (HIC), hydroxyapatite (HA) chromatography, and size exclusion chromatography (SEC). In some embodiments including a surge vessel, e.g., a first disposable surge vessel (SUSV1), upstream of the first chromatography system and fluidly connected to the first chromatography system, there is an automatic control device including a detector for measuring the fluid volume of the surge vessel, e.g., SUSV1, and a processor for varying the pump speed of the first chromatography system to maintain a preset volume range of the surge vessel (e.g., SUSV1). The volume of SUSV1 is typically about 200 L, but can be set smaller or larger depending on the process flow rate and desired residence time (which impacts the time frame in which one can react to process perturbations). Operation of the first chromatography system collects or captures the protein of interest in the protein isolate fraction.

[0255] The first chromatography system, the second chromatography system, and / or the optional third chromatography system can be configured with one, two, three or more different chromatography matrices (e.g., chromatography columns) fluidly connected, preferably in series, as needed for the protein of interest, and optionally arranged on a mobile, replaceable, or disposable disposable unit, skid, or "cart."

[0256] In some embodiments of the invention, the second chromatographic system includes a disposable membrane adsorber (MA), such as a surface-functionalized membrane. Such membrane adsorber can include anion exchange groups for mAb polishing operation in negative mode, where trace impurities are removed without binding to the protein of interest (so-called "flow-through chromatography"). Examples include, but are not limited to, Sartobind® Q or Sartobind STIC® (Sartorius Stedim Biotech), or Mustang® Q (Pall Life Sciences), or NatriFlo® HD-Q (Natrix Separations). Alternatively, the membrane adsorber can include cation exchange groups, such as Sartobind® S (Sartorius Stedim Biotech), or Mustang® S (Pall Life Sciences), or Natrix® HD-Sb (Natrix Separations). In some embodiments, membranes with other functional groups can be used to perform hydrophobic interaction chromatography (HIC).

[0257] An embodiment of the process (and automated equipment) of the present invention includes subsequently switching the protein isolate fraction obtained or collected from the first chromatography system to a low pH or detergent viral inactivation system and neutralization system (i.e., if neutralization is required following low pH viral inactivation) to obtain a viral inactivation product pool that contains the protein of interest (e.g., but is not limited to, a protein drug). However, optionally, the protein isolate fraction may be fluidly switched from the first chromatography system to a low pH or detergent viral inactivation system before the protein isolate fraction is fluidly fed to the low pH or detergent viral inactivation system. (i) A second disposable surge container; or (ii) capable of feeding either of at least two automatically switchable alternative disposable collection vessels (SUCV1 and SUCV2): (i) a disposable surge vessel, or (ii) SUCV1 and SUCV2 adapted to receive the protein isolate fraction from the first chromatography system and feed the protein isolate fraction to a low pH or detergent viral inactivation system.

[0258] In an alternative embodiment, the low pH or detergent viral inactivation system, and optionally the neutralization system (i.e., if neutralization is required following low pH viral inactivation), comprises: (i) A (third) disposable surge receptacle; or (ii) at least two automatically switchable alternative disposable collection vessels (SUCV1 and SUCV2), (i) a disposable surge vessel, or (ii) SUCV1 and SUCV2 contained in a low pH or detergent viral inactivation system and optionally a neutralization system, adapted to receive the protein isolate fraction from the first chromatography system.

[0259] The volumes of SUSV2 and SUCV1 and SUCV2 are typically about 100 L each, but this can be smaller or larger depending on how often the pools are further processed. For example, for an elution pool of about 20-25 L, a 50 L vessel was effectively used as SUCV1 and SUCV2. After using the low pH virus inactivation system, a neutralization system is required to restore the isolated protein in solution to a near neutral pH. The term "low pH" refers to a pH value of about pH 3.7 or less at which the protein isolate fraction is held (typically for at least 30-90 minutes) to inactivate contaminating virus particles. (See, e.g., Chinniah, S et al., Characterization of operating parameters for XMuLV inactivation by low pH treatment, Biotechnol Prog. 32(1):89-97 (2016)). When a detergent (e.g., Triton-X-100 and / or tri(n-butyl)phosphate ("TNBP")) viral inactivation system is used, treatment of the protein isolate fraction with a neutralization system is not usually necessary, unless less than neutral pH conditions are also used, which would prevent further effective purification or stable storage of the viral inactivation product pool. (See, e.g., Dichtelmuller et al., Effective virus inactivation and removal by steps of Biotest Pharmaceuticals, Results in Immunology 2:19-24(2012); Ellgard et al., Evaluation of the virus clearance capacity and robustness of the manufacturing process for the recombinant factor VIII protein, turoctocog alfa IGIV production process, Protein Expression and Purification 129:94-100(2017)).

[0260] The resulting viral inactivation product pool is then introduced into a second chromatography system (in some embodiments after being stored for at least 10 days, or at least 20 days, or at least 30 days) in a temperature-controlled or chilled holding vessel (HV1) to obtain a purified product pool containing the protein of interest. The second chromatography system can be configured with one, two, three or more different chromatography matrices (e.g., chromatography columns) preferably fluidly linked in series as needed for further purification of the protein of interest, and optionally disposed on a mobile, replaceable or disposable disposable unit, skid or "cart".

[0261] Introducing the viral inactivation product pool into the second chromatography system is optionally controlled according to a coordinated schedule with respect to the culturing step and the viral inactivation step. The coordinated schedule is calculated to maximize efficient routing of the viral inactivation product pool into the second chromatography system. This loading of the viral inactivation product pool into the second chromatography system according to the coordinated schedule is automatic (continuous mode) or by batch-wise manual control (semi-continuous mode). (See also Garcia, FA and Vandiver, MW, Throughput Optimization of Continuous Biopharmaceutical Manufacturing Facilities, PDA J Pharm Sci Technol 71(3):189-205(2017)).

[0262] From the second chromatography system, the resulting purified product pool containing the protein of interest is fluidically switched to an optional third chromatography system and / or virus filtration system to obtain a virus-free filtrate containing the protein. The switching of the purified product pool to the optional chromatography system and / or virus filtration system is by automatic or manual control. The optional third chromatography system is configured with one, two, three or more different chromatography matrices (e.g., chromatography columns) preferably fluidly connected in series as needed for further purification of the protein of interest, and can be optionally arranged on a mobile, replaceable or disposable disposable unit, skid or "cart". If a third chromatography system is not used in the process (or equipment) of the present invention, the purified product pool is switched and fluidically flows directly to the virus filtration system. Useful virus systems, including disposable virus filtration systems, are commercially available.

[0263] The resulting virus-free filtrate is then fluidly switched to an UF / DF system to obtain a composition comprising a purified protein of interest (e.g., a purified protein drug substance). The switching of the virus-free filtrate to the UF / DF system can be automatic or manually controlled.

[0264] Useful examples of ultrafiltration / diafiltration systems include ultrafiltration cassettes, including, but not limited to, Pellicon® 3 Ultracel 30 kDa membrane (Millipore Sigma), Sartocon® ECO Hydrosart® 30-kDa regenerated cellulose membrane (Sartorius), and Delta 30-kDa regenerated cellulose membrane (Pall Biotech).

[0265] At the end of the process, the purified protein (e.g., protein drug substance) can be stored in a sterile container, such as, but not limited to, a disposable sterile container (e.g., Celsius®-FFT system, Sartorius), a ready-to-use carboy, or can be directly processed into a formulation.

[0266] In some embodiments of the process (and automated equipment) of the present invention, one or more of the first chromatography system, the second chromatography system, the third chromatography system, the low pH or detergent viral inactivation system, the neutralization system, the viral filtration system, or the UF / DF system comprise disposable components. The use of disposable components provides efficiency, safety, and reduces the ultimate cost of performing the process of the present invention. Furthermore, in a scenario where multiple disposable perfusion bioreactors are utilized in a facility for the production of purified proteins of interest (such as, but not limited to, purified protein drug substances), multiple operations performed on each bioreactor can be performed simultaneously. For example, an UF / Diafiltration operation can be performed on the virus-free filtrate from the first perfusion bioreactor, while a chromatography operation can be performed on the virus-inactivated product pool generated by the virus inactivation system (and, optionally, the neutralization system) that processes the protein isolate fraction received after the treatment by the first chromatography system of the cell-free permeate from the cultivation in the second disposable perfusion bioreactor. In another example, an UF / Diafiltration operation can be performed on the virus-free filtrate finally generated by the method of the invention from the cultivation in the first disposable perfusion bioreactor, while a virus filtration operation can be performed on the virus-inactivated product pool finally generated by the method of the invention from the cultivation in the second perfusion bioreactor. In a further embodiment, at least one chromatography process and / or viral filtration process performed on the virus-free filtrate produced from the first perfusion bioreactor can be performed during a continuous chromatography capture or viral inactivation process performed on the cell-free permeate volume produced by the second disposable bioreactor according to the process of the invention.

[0267] Purity of Water and Other Components. Water and all other components used in the steps of the process of the invention to express, purify and manufacture formulations of purified drug substances are preferably at purity levels that meet the applicable legal or pharmacopoeial standards required for such pharmaceutical compositions and drug products in the jurisdiction of interest, such as, for example, the United States Pharmacopoeia (USP), the European Pharmacopoeia, the Japanese Pharmacopoeia, or the Chinese Pharmacopoeia. For example, according to the USP, water for injection is used as an excipient in the manufacture of parenteral and other formulations where the endotoxin content of the product must be controlled, as well as in other pharmaceutical applications such as washing certain equipment and components that come into contact with parenteral products, and the minimum quality of the source or feed water for generating water for injection is drinking water as defined by the US Environmental Protection Agency (EPA), the EU, Japan, or the WHO.

[0268] Automation and Control Systems

[0269] Conventional production facility control systems are typically designed to control preset configurations of the facility. In these scenarios, the logical and hardware couplings between the equipment do not change. Thus, the identifiers and control actions that can be performed for each equipment are static. The implementation of the production facility control system described herein supports variable configurations of equipment in the production line with respect to the automated facilities and processes of the present invention for manufacturing purified proteins of interest (such as, but not limited to, protein drug substances). In these situations, the equipment may have different functions, perform different operations, and / or be controlled using different sets of control commands and / or variables based on the location of the equipment in the production line. Thus, the production line and control system described herein include different software configurations and physical hardware than conventional systems. The ability to configure a production line in an automated facility using the same group of control modules with different configurations of equipment on the production line may be an implementation of the so-called "FlexTrain" automation.

[0270] The embodiments described herein can be performed by one or more systems that can automatically control the flow of materials through each step of the process to produce a protein of interest, such as, but not limited to, a protein drug substance. Alternatively, at least some of the control functions can be performed by operator intervention, and there can be circumstances (particularly process interruptions) that may require operator intervention. The control functions can be performed using process data obtained from sensors coupled to various equipment used to produce the purified protein of interest. The sensors can include temperature sensors, pH sensors, flow sensors, weight sensors (e.g., load cells), volume sensors (e.g., guided wave radar sensors), pressure sensors, timers, capacitance sensors, optical density sensors, or combinations thereof. The data generated by the sensors can be collected locally by the equipment. In certain embodiments, the equipment can transfer sensor data to a production facility control system. The production facility control system can collect data from sensors on several pieces of equipment used to manufacture the purified protein of interest (e.g., purified protein drug substance). The production facility control system can include one or more computing devices and / or one or more data stores in electronic communication with each other. In some scenarios, at least a portion of the one or more computing devices and / or one or more data stores may be co-located. Additionally, at least a portion of the one or more computing devices and / or one or more data stores may be located remotely from equipment included in the production facility. In this situation, at least a portion of the operations performed by the production facility control system may be implemented in a cloud computing architecture.

[0271] The data collected from the sensors can be stored in an electronic data store, which may be referred to herein as a "data historian." In various embodiments, a first data historian can collect and store data for at least a subset of the equipment operating at the purified protein production facility (e.g., for the production of purified protein drug substance or other protein of interest). The first data historian can store the data for a period of time and then forward the data to a second data historian, which is a repository of data collected regarding the operation of the equipment coupled to the production facility control system. The DeltaV historian and / or Pi historian are examples of redundant data historian systems commonly used in commercial manufacturing plants for protein drug substances. In certain circumstances, the first data historian can then be reset and begin collecting and storing additional data from the purified protein production facility (e.g., for the manufacture of purified protein drug substance) for an additional period of time. The production facility control system can also include one or more batch historians that collect and store data related to the operation of the equipment included in the production facility to produce a particular batch of purified protein of interest (e.g., but not limited to, protein drug substance). The data historian may be accessed by the production facility control system and analyzed to determine parameters for operation of equipment included under the control of the production facility control system.

[0272] The production facility control system can analyze data obtained from the sensors and determine the operating conditions of one or more pieces of equipment. In some cases, set points and acceptable operating parameters for the operation of the equipment, and / or run recipes can be entered into the system by an operator. In other situations, set points and acceptable operating parameters for the operation of the equipment, and / or run recipes can be automatically sent to one or more pieces of equipment utilized in the purified protein production line (e.g., for the manufacture of purified protein drug substance). Alerts and alarm notifications can also be generated based on the sensor data. For example, in a situation where the sensor data indicates that the operating conditions of an equipment in the purified protein production line are outside of a threshold range, the system can trigger an alarm and send a notification to an operator.

[0273] Various equipment used to produce a purified protein of interest (e.g., purified protein drug substance) can include one or more communication interfaces that allow communication between the equipment and / or with a production facility control system. In some embodiments, the production facility control system can operate as a process automation system (PAS). The communication interface can include hardware devices, firmware devices, and / or software implemented systems that allow communication of data between the equipment used in the purified protein production line and / or with the production facility control system. The communication interface can allow communication of data over a number of networks, such as a local area wired network, a local area wireless network, a wide area wireless network, and / or a wide area wired network. In certain examples, the communication interface can include an Ethernet network communication interface, an Internet Protocol network communication interface, an Institute of Electrical and Electronics Engineers (IEEE) 802.11 wireless network communication interface, a Bluetooth communication interface, or a combination thereof.

[0274] The equipment used to produce purified proteins of interest (such as, but not limited to, protein drug substances) can include one or more processors and one or more memory devices. The one or more processors can be central processing units, such as standard programmable processors, that perform arithmetic and logical operations necessary for the operation of the computing system. The one or more memory devices can include volatile and non-volatile memory and / or removable and non-removable media implemented in any type of technology for storing information, such as computer-readable instructions, data structures, program modules, or other data. Such computer-readable storage media can include, but are not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disk (DVD) or other optical storage, magnetic cassettes, magnetic tape, solid state storage, magnetic disk storage, RAID storage systems, storage arrays, network attached storage, storage area networks, cloud storage, removable storage media, or any other media that can be used to store desired information and that can be accessed by the production facility control system or the individual equipment included in the purified protein production line.

[0275] According to the automated equipment and process of the present invention for manufacturing a purified protein of interest (such as, but not limited to, a protein drug substance), at least some of the equipment included in the purified protein production line and the production equipment control system can store one or more modules that can be executed to control the operation of the equipment included in the purified protein production line. The modules can include computer readable instructions that can be executed to cause the equipment included in the purified protein production line to perform one or more operations. The modules can be part of a framework that enables the equipment included in the purified protein production line to produce the purified protein (e.g., purified protein drug substance) continuously or semi-continuously. The operations performed by the various equipment included in the purified protein production line can be related to a start-up process, a holding process, a stopping process, a feeding process, or the end of the production process.

[0276] In certain embodiments, the control system described herein can be used to control a production line having flexible configurations. That is, the control system described herein can accommodate multiple configurations utilizing portable equipment that can be coupled to other components of the production line. In various embodiments, the production line can include one or more skids that include original manufacturer equipment, such as a disposable bioreactor system, a perfusion system, or a continuous chromatography system. The skid can also include flow control devices, such as pumps. Additionally, the skid can include one or more communication interfaces that allow for the portable equipment, also referred to herein as "drops," to be physically coupled to the skid. The physical coupling between the portable equipment and the skid can be accomplished using an electrical cable. The electrical cable can be configured to enable Ethernet communication. In a particular example, the electrical cable can be a Recommended Standard 232 (RS-232) cable.

[0277] The portable equipment may include or otherwise be coupled to a network gateway hardware device that enables communication between each portable equipment and the production facility control system. The network gateway hardware device of each portable equipment may be coupled to a communication interface of a respective skid. Further, at least some of the skids may be logically configured to be coupled to various portable equipment. In this manner, the portable equipment may be physically connected to a particular skid based on the configuration of a particular production line, and the skids may be configured to operate in different configurations based on the different equipment coupled to the skid.

[0278] Additionally, the portable devices may be coupled to at least one communication and / or storage device, such as a dongle. The communication and / or storage device may store information provided to each device to which it is coupled that enables control of the respective device via the production facility control system. The communication and / or storage device may store information including one or more identifiers of the respective device, one or more functions of the respective device, one or more control signals corresponding to the respective device, one or more status flags associated with the respective device, or combinations thereof. In some examples, the data stored by the communication and / or storage device may be based at least in part on the function or type of the respective device. In situations where the portable devices are located at different locations along the production line and / or have different capabilities, the communication and / or storage device of the portable device may switch to additional communication and / or storage devices indicating different functions and different identifiers of the portable devices.

[0279] Additionally, the control systems described herein may include additional logical layers that may be used on top of conventional control software and systems. In certain implementations, the control systems described herein may include additional abstraction layers that allow for the assignment, also referred to as "binding," of portable equipment to various identifiers, tags, operating conditions, and flags that correspond to a particular set of functions for a particular piece of equipment at a particular location along a production line. In this manner, equipment is not logically represented within the control system until the equipment's location and functions are known. Thus, portable equipment may be coupled with the skid in various combinations without the need to modify the underlying control software utilized to control the components of the skid and to control the portable equipment.

[0280] In an illustrative example, an automated equipment production line of the present invention for manufacturing a purified protein of interest (such as, but not limited to, a purified protein drug substance) can include a first skid including a disposable bioreactor system, a second skid including a perfusion system, and a third skid including a continuous first chromatography system. The skid can be configured to couple to a portable piece of equipment. For example, the skid can include interfaces and physical hardware for coupling to a portable mixing tank, a filter bank, a storage container, a surge receptacle, a holding receptacle, a diverter valve system (for switching between automatically switchable alternative dual or multi-path unit operations, e.g., SUCV1 and SUCV2), and / or other flow control devices. The mixing tank (or interchangeably, a "mixing receptacle") or part of the storage receptacle can function as a feed tank or collection receptacle that can function as a surge receptacle in a continuous or semi-continuous format manufacturing process, or as a holding receptacle in a batch mode format manufacturing process.

[0281] After coupling the portable equipment to the skid, the portable equipment can be registered with the production facility control system. The portable equipment can have a unique address that allows the portable equipment to communicate to the production facility control system. The unique address can indicate the type and unit identifier of the portable equipment to the production facility control system. The dongle coupled to the portable equipment can store additional identifiers corresponding to the location of the skid to which the portable equipment is coupled and one or more functional roles of the portable equipment. For example, a mixing tank can be identified as a feed tank or a collection tank based on the location of the portable equipment and the logical association of the drop to which the portable equipment is coupled. In another example, a filter bank can be identified as a virus filtration device in a first configuration of the production line and then as a diafiltration device in a second configuration of the production line. In these situations, a first dongle can be coupled to the filter bank of the first configuration of the production line and a second dongle can be coupled to the filter bank of the second configuration of the production line. Additionally, the type of filter used in the filter bank can be changed when the filter bank is used at a different location in the production line.

[0282] In response to obtaining information from the portable equipment after it is coupled to the skid, the production facility control system can determine the location and function of the portable equipment and assign a corresponding control template to the portable equipment. For example, in a situation where the mixing tank is functioning as a collection tank, the production facility control system can assign a first set of tags, flags, identifiers, and set points to the mixing tank, and in a situation where the mixing tank is functioning as a feed tank, the production facility control system can assign a second set of tags, flags, identifiers, and set points to the mixing tank. The production facility control system can then assign a particular set of control modules to the portable equipment based on the information obtained from the portable equipment after it is coupled to the skid.

[0283] In various embodiments, equipment that is not considered to be portable, such as large collection tanks, may also be coupled to the skid. In these scenarios, the non-portable equipment may not include hardware and / or communications and storage that allows for dynamic configuration of the non-portable equipment to the production facility control system. If the non-portable equipment is not configured for dynamic configuration, an operator of the production facility control system may manually establish the templates and / or control modules used to control the operation of the non-portable equipment.

[0284] In addition to controlling the equipment included in the production line, the production facility control system can also track the decay rate of batches during the production of a purified protein of interest (e.g., but not limited to, a purified protein drug substance). A "decay rate" is the period during which materials used in the production of a sublot can be identified and tracked. For example, materials (e.g., buffers, cell culture media, etc.) used in the resulting chromatography step eluate pool collection, of which there may be many, can be dynamically identified and tracked by "decay rate". In a continuous batch production process, the production facility control system can estimate the decay rate of the purified protein production process. In various embodiments, the production facility control system can assign a batch identifier to a particular portion of the production of a batch and initiate a decay monitor until the current batch identifier is changed to a new batch identifier and a new decay monitor is performed for the new batch identifier.

[0285] In an illustrative example, the production equipment control system can determine that the filter bank is coupled between the perfusion bioreactor and the first chromatography system based on information obtained from a dongle coupled to the filter bank. In these circumstances, the filter bank can operate as a depth filter. The production equipment control system can identify one or more control modules, flags, and / or status identifiers of the depth filter and execute one or more control modules while the filter bank is in use in the production line. The production equipment control system can monitor the pressure in the filter assembly of the filter bank based on a pressure value obtained from a pressure sensor included in the filter assembly. The production equipment control system can determine that the pressure in the first filter assembly through which the material is flowing has reached at least a threshold level. The threshold level of pressure can indicate that the filter included in the first assembly needs to be replaced due to a reduction in the amount of material that can be processed by the filter. The production equipment control system can then send a signal to control a diverter valve coupled to the filter bank to flow the material through a second filter assembly of the filter bank. The filter included in the first filter assembly can then be replaced.

[0286] By way of further example, the following embodiments of the invention are recited.

[0287] Embodiment 1: A process for producing a purified protein of interest, comprising the steps of:

[0288] (a) culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that permit the cells to secrete a protein into the liquid medium over a production culture period of at least 10 days, wherein during the production culture period, fresh sterile liquid medium is periodically or continuously added to the one or more disposable perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors in direct relation to a volume of culture that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed, and the volume of permeate removed is automatically and fluidly delivered from the one or more disposable perfusion bioreactors to a disposable surge vessel and then to a first chromatography system, whereby the protein is collected in a protein isolate fraction.

[0289] Embodiment 2: A process for producing a purified protein of interest, comprising the steps of:

[0290] (a) culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that permit the cells to secrete a protein into the liquid medium over a production culture period of at least 10 days, wherein during the production culture period, fresh sterile liquid medium that is simultaneously mixed from a plurality of different concentrated medium component solutions and an aqueous diluent is periodically or continuously added to the one or more disposable perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors in direct relation to a volume of culture that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed, and wherein the volume of permeate removed is automatically and fluidly delivered from the one or more disposable perfusion bioreactors to a disposable surge vessel and then to a first chromatography system, whereby the protein is collected in a protein isolate fraction.

[0291] Embodiment 3: The process of embodiment 1 to 2, further comprising the steps of:

[0292] (b) switching the protein isolate fraction to a low pH or detergent viral inactivation system, and optionally a neutralization system, to obtain a viral inactivation product pool containing the protein.

[0293] Embodiment 4: The process of embodiments 1 to 3, further comprising the steps of:

[0294] (c) introducing the viral inactivation product pool into a second chromatography system to obtain a purified product pool comprising the protein;

[0295] (d) switching the purified product pool containing the protein to an optional third chromatography system and / or a viral filtration system to obtain a virus-free filtrate containing the protein; and

[0296] (e) switching the virus-free filtrate through an ultrafiltration / diafiltration system to obtain a composition containing the purified protein of interest.

[0297] Embodiment 5: The process according to any of embodiments 1 to 4, wherein the protein of interest is a recombinant protein.

[0298] Embodiment 6: The process of any of embodiments 1 to 5, wherein the protein of interest is a therapeutic protein (or other medically useful protein).

[0299] Embodiment 7: The process of any of embodiments 1 to 6, wherein one or more of the first chromatography system, the second chromatography system, the third chromatography system, the low pH or detergent viral inactivation system, the neutralization system, the viral filtration system, or the ultrafiltration / diafiltration system comprises one or more disposable components.

[0300] Embodiment 8: The process according to any of embodiments 1 to 7, wherein the mammalian cells are cultured in 2, 3, 4, 5 or 6 disposable perfusion bioreactors.

[0301] Embodiment 9: The process of any of embodiments 1 to 8, wherein the one or more disposable bioreactors can contain a volume of liquid medium from about 50 L to about 4000 L.

[0302] Embodiment 10: The process of any of embodiments 2 to 9, wherein fresh sterile liquid medium is added to one or more perfusion bioreactors by directly injecting the different concentrated medium component solutions in fixed ratios relative to each other into the perfusion bioreactor, while an aqueous diluent is also added in varying ratios relative to the different concentrated component solutions to maintain a constant culture volume in each perfusion bioreactor.

[0303] Embodiment 11: The process of any of embodiments 2 to 9, wherein fresh sterile liquid medium is added to one or more perfusion bioreactors by injecting a plurality of different concentrated medium component solutions and an aqueous diluent in fixed ratios relative to one another directly into the perfusion bioreactors to maintain a constant culture volume in each perfusion bioreactor.

[0304] Embodiment 12: The process of any of embodiments 2 to 9, wherein fresh sterile liquid medium is added to one or more perfusion bioreactors by injecting a plurality of different concentrated medium component solutions and an aqueous diluent in a fixed ratio relative to each other into a mixing chamber, where fresh sterile liquid medium is mixed simultaneously and then added to each perfusion bioreactor to maintain the culture volume constant.

[0305] Embodiment 13: A process according to any of embodiments 1 to 12, wherein an automatic control device including a detector is used to measure the fluid volume in the disposable surge receptacle, and the processor varies the pump speed of the first chromatography system to maintain a preset volume range in the disposable surge receptacle.

[0306] Embodiment 14: The process of any of embodiments 3 to 13, wherein one or more of steps (b), (c), (d), or (e) are performed automatically and fluidly with uninterrupted flow from the previous step, a surge receptacle is used during one or more steps, and the processor varies the pump speed in the subsequent step to adjust the preset volume range of the surge receptacle preceding the subsequent step.

[0307] Embodiment 15: The process of any of embodiments 3 to 14, wherein an in-line or in-vessel adjustment of pH and / or conductivity loading is performed between one or more of steps (b), (c), (d), or (e).

[0308] Embodiment 16: The process of any one of embodiments 1 to 15, comprising:

[0309] (i) a process automation system in electronic communication with at least one or more of the disposable perfusion bioreactor, the disposable surge vessel, and the first chromatography system;

[0310] (ii) the process automation system stores a first set of control modules for controlling operation of at least one single-use perfusion bioreactor of the one or more single-use perfusion bioreactors;

[0311] (iii) the process automation system stores a second set of control modules for controlling operation of the supply tank;

[0312] (iv) the process automation system stores a third set of control modules for controlling operation of the collection tank;

[0313] (v) The process, wherein at least one disposable perfusion bioreactor is logically configured to be coupled to one or more feed tanks, one or more harvest tanks, or a filter bank.

[0314] Embodiment 17: The process of embodiment 16, wherein the at least one disposable perfusion bioreactor is disposed on a skid, and the skid comprises a plurality of communication interfaces for electronically coupling the at least one disposable perfusion bioreactor to a plurality of portable instruments.

[0315] Embodiment 18: The process of embodiment 17, further comprising:

[0316] (vi) determining, by the process automation system, that a disposable surge receptacle is coupled to one of the plurality of communication interfaces based on data received via the communication interface, the data indicating an identifier of the disposable surge receptacle and a function of the disposable surge receptacle; and

[0317] (vii) determining, based at least in part on the identifier and functionality of the disposable surge receptacle, that the disposable surge receptacle is a collection tank and that the third set of control modules is for controlling operation of the disposable surge receptacle.

[0318] Embodiment 19: The process of embodiment 18, further comprising:

[0319] (viii) determining, by the process automation system, that the mixing vessel is coupled to an additional communication interface of the plurality of communication interfaces based on additional data received via the additional communication interface, the additional data indicating an additional identifier of the mixing vessel and an additional function of the mixing vessel; and

[0320] (ix) determining, based at least in part on the additional identifier and the additional functionality of the mixing vessel, that the mixing vessel is a supply tank and that the second set of control modules is for controlling operation of the mixing vessel;

[0321] Embodiment 20: A process described in any of embodiments 18 to 19, wherein an identifier of the disposable surge receptacle and the functions of the disposable surge receptacle are stored in a dongle coupled to the disposable surge receptacle.

[0322] Embodiment 21: The process of any of embodiments 1 to 20, wherein the production culture period is at least 20 days.

[0323] Embodiment 22: An automated system for producing a purified protein of interest, comprising:

[0324] (a) one or more disposable perfusion bioreactors capable of containing a liquid medium under conditions that permit the cultured cells to secrete protein into the liquid medium for a production culture period of at least 10 days, the disposable perfusion bioreactors being adapted to fluidly receive fresh sterile liquid medium into each of the perfusion bioreactors in direct relation to the volume of conditioned medium that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed during the production culture period;

[0325] (b) a first disposable surge vessel (SUSV1) into which the removed permeate volume is automatically and fluidly delivered from the one or more disposable perfusion bioreactors; and

[0326] (c) a first chromatography system adapted to automatically and fluidly receive the cell-free permeate from SUSV1, whereby proteins are captured in a protein isolate fraction;

[0327] Automated equipment controlled by a process automation system (PAS).

[0328] Embodiment 23: The automated equipment of embodiment 22, further comprising a plurality of reservoirs each adapted to contain a concentrated medium component solution or an aqueous diluent, the plurality of reservoirs being fluidly connected to the perfusion bioreactor either directly or indirectly via an optional mixing vessel adapted to receive the concentrated medium component solution and the aqueous diluent in a predetermined ratio from the plurality of reservoirs and mix them simultaneously, the optional mixing vessel being fluidly connected directly to the perfusion bioreactor.

[0329] Embodiment 24: The automated system of embodiment 22 to 23, further comprising:

[0330] (d) a low pH or detergent viral inactivation system, and optionally a neutralization system, adapted to automatically and fluidly receive the protein isolate fraction from the first chromatography system, thereby obtaining a viral inactivation product pool comprising the protein; and

[0331] (e) a holding vessel or a second disposable surge vessel adapted to receive the viral inactivation product pool.

[0332] Embodiment 25: The automated system of embodiment 22 to 24, further comprising:

[0333] (f) a second chromatography system adapted to fluidly receive the viral inactivation product pool from the holding vessel or the second disposable surge vessel, thereby obtaining a purified product pool comprising the protein;

[0334] (g) an optional third chromatography system and / or virus filtration system adapted to fluidly receive the purified product pool comprising the protein from the second chromatography system, thereby obtaining a virus-free filtrate comprising the protein; and

[0335] (h) An ultrafiltration / diafiltration system adapted to fluidly receive the virus-free filtrate from the second chromatography system, or from the third chromatography system and / or the viral filtration system, thereby obtaining a purified protein of interest.

[0336] Embodiment 26: The automated equipment according to any of embodiments 22 to 25, wherein the one or more disposable perfusion bioreactors can contain a liquid medium volume of about 50 L to about 4000 L.

[0337] Embodiment 27: An automated equipment described in any of embodiments 22 to 26, further comprising an automatic control device including a detector for measuring the fluid volume of SUSV1 and a processor for changing the pump speed of the first chromatography system to maintain a preset volume range of SUSV1.

[0338] Embodiment 28: The automated equipment of any of embodiments 24 to 27, wherein one or more of the first chromatography system, the second chromatography system, the third chromatography system, the low pH or detergent viral inactivation system, the neutralization system, the viral filtration system, or the ultrafiltration / diafiltration system comprise disposable components.

[0339] Embodiment 29: A method for detecting a chromatographic system comprising:

[0340] (i) A second disposable surge container; or

[0341] (ii) at least two automatically switchable alternative disposable collection vessels (SUCV1 and SUCV2) adapted to receive protein isolate fractions;

[0342] 29. The automated equipment of any of embodiments 24 to 28, wherein (i) and (ii) are adapted to receive the protein isolate fraction from the first chromatography system and fluidly feed the protein isolate fraction to a low pH or detergent viral inactivation system.

[0343] Embodiment 30: A low pH or detergent viral inactivation system, and optionally a neutralization system, comprising:

[0344] (i) a second disposable surge vessel adapted to receive the protein isolate fraction; or

[0345] (ii) at least two automatically switchable alternative disposable collection vessels (SUCV1 and SUCV2) adapted to receive protein isolate fractions;

[0346] 29. The automated equipment according to any of embodiments 24 to 28, wherein viral inactivation, and optionally neutralization, is carried out in a second disposable surge vessel, or in SUCV1 and SUCV2.

[0347] Embodiment 31: An automated equipment described in any of embodiments 22 to 30, further comprising a hollow fiber membrane, a series of depth filters, or a filtration cart before the permeate is automatically and fluidly supplied to SUSV1.

[0348] Embodiment 32: The automated equipment of any of embodiments 22 to 31, comprising in (e) a disposable surge vessel adapted to receive the viral inactivation product pool.

[0349] Embodiment 33: An automated equipment described in any of embodiments 22 to 32, further comprising a heat exchanger upstream of SUSV1.

[0350] Embodiment 34: An automated equipment described in any of embodiments 22 to 33, further comprising a filtration system upstream of SUSV1.

[0351] Embodiment 35: The automated equipment according to any one of embodiments 24 to 34,

[0352] (i) a second chromatography system;

[0353] (ii) an optional third chromatographic system;

[0354] (iii) viral filtration systems; and

[0355] (iv) one or more of the ultrafiltration / diafiltration systems

[0356] An automated installation that is automatically and fluidly connected to a preceding system, and in which a surge receptacle is optionally used to regulate the uninterrupted flow of material between the connected systems.

[0357] Embodiment 36: The automated equipment according to any one of embodiments 22 to 35,

[0358] The first equipment includes at least one or more disposable perfusion bioreactors, SUSV1, a first chromatography system, a low pH or detergent viral inactivation system, a holding vessel or disposable surge vessel, a second chromatography system, an optional third chromatography system and / or a viral filtration system, and an UF / DF system arranged in a first configuration of a production line for a purified protein of interest;

[0359] An automated facility, wherein a first plurality of control modules are implemented to control the operation of a first piece of equipment.

[0360] Embodiment 37: The automated equipment according to embodiment 36, comprising:

[0361] a second piece of equipment is disposed in a second configuration of a production line for additional purified proteins of interest, the second configuration of the production line including at least one or more disposable perfusion bioreactors, a first chromatography system, a low pH or detergent viral inactivation system, a second chromatography system, an UF / DF system, and a plurality of mixing vessels;

[0362] the second configuration of the production line is different from the first configuration of the production line;

[0363] a second plurality of control modules implemented for controlling operation of a second device;

[0364] At least one mixing vessel of the plurality of mixing vessels is included in both the first configuration and the second configuration;

[0365] 1. An automated system, wherein at least one mixing vessel has a first function in a first configuration and a second function in a second configuration, the second function being different from the first function.

[0366] Embodiment 38: A portable filter bank including a plurality of filter assemblies,

[0367] a portable filter bank, wherein a first filter assembly of the plurality of filter assemblies includes a first filter and a second filter assembly of the plurality of filter assemblies includes a second filter;

[0368] A production facility control system, comprising:

[0369] monitoring a pressure within the first filter assembly as material flows through the first filter assembly;

[0370] Determining that the pressure in the first filter assembly is at least a threshold value;

[0371] 38. The automated equipment of any of embodiments 22 to 37, further comprising a production equipment control system that sends a signal to operate a diverter valve coupled to the first filter assembly and the second filter assembly to cause the material to flow into the second filter assembly.

[0372] Embodiment 39: The automated equipment of embodiment 38, wherein during a first period of time, the filter bank is coupled between a first disposable surge vessel (SUSV1) and one or more disposable perfusion bioreactors, the material comprises permeate, and the filter bank is coupled to a first dongle indicating a first identifier of the filter bank and a first function of the filter bank.

[0373] Embodiment 40: The automated equipment of embodiment 39, wherein during a second period of time, the filter bank is included in a low pH or detergent viral inactivation system, the material is a virus-free filtrate, and the filter bank is coupled to a second dongle indicating a second identifier of the filter bank and a second function of the filter bank.

[0374] Embodiment 41: The automated equipment of any of embodiments 22 to 40, wherein the one or more disposable perfusion bioreactors can contain liquid medium under conditions that allow the cultured cells to secrete proteins into the medium for a production culture period of at least 20 days.

[0375] Embodiment 42: The process according to any one of embodiments 1 to 21 or the automated installation according to any one of embodiments 22 to 41, wherein the protein of interest is a recombinant protein and / or a therapeutic protein.

[0376] Embodiment 43: A process for producing a purified protein drug substance comprising a protein of interest, the process comprising the steps of:

[0377] (a) culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that permit the cells to secrete a protein into the liquid medium over a production culture period of at least 10 days, wherein during the production culture period, fresh sterile liquid medium is periodically or continuously added to the one or more disposable perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors in direct relation to a volume of culture that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed, and the volume of permeate removed is automatically and fluidly delivered from the one or more disposable perfusion bioreactors to a disposable surge vessel and then to a first chromatography system, whereby said protein is collected in a protein isolate fraction;

[0378] (b) switching the protein isolate fraction to a low pH or detergent viral inactivation system, and optionally a neutralization system, to obtain a viral inactivation product pool containing the protein;

[0379] (c) introducing the viral inactivation product pool into a second chromatography system to obtain a purified product pool comprising the protein;

[0380] (d) switching the purified product pool containing the protein to an optional third chromatography system and / or a viral filtration system to obtain a virus-free filtrate containing the protein; and

[0381] (e) Switching the virus-free filtrate into an ultrafiltration / diafiltration system to obtain purified protein drug substance containing the protein of interest.

[0382] Embodiment 44: The process of any of embodiments 42 to 43, wherein fresh sterile liquid medium is mixed simultaneously from a plurality of different concentrated medium component solutions and an aqueous diluent and then added to one or more perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors.

[0383] Embodiment 45: An automated facility for producing a purified protein drug substance, comprising:

[0384] (a) one or more disposable perfusion bioreactors capable of containing a liquid culture medium under conditions that permit cultured mammalian cells to secrete a protein of interest into the medium over a production culture period of at least 10 days, the disposable perfusion bioreactors being adapted to fluidly receive fresh sterile liquid culture medium into each of the perfusion bioreactors in direct relation to the volume of conditioned medium that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed during the production culture period;

[0385] (b) a first disposable surge vessel (SUSV1) into which the removed volume of permeate is automatically and fluidly supplied from one or more disposable perfusion bioreactors;

[0386] (c) a first chromatography system adapted to automatically and fluidly receive the permeate from SUSV1, whereby the protein is captured in a protein isolate fraction;

[0387] (d) a low pH or detergent viral inactivation system, and optionally a neutralization system, adapted to automatically and fluidly receive the protein isolate fraction from the first chromatography system, thereby obtaining a viral inactivation product pool comprising the protein;

[0388] (e) a holding vessel or disposable surge vessel adapted to receive the viral inactivation product pool;

[0389] (f) a second chromatography system adapted to fluidly receive the viral inactivation product pool from the holding vessel or the disposable surge vessel, thereby obtaining a purified product pool comprising the protein;

[0390] (g) an optional third chromatography system and / or a viral filtration system adapted to fluidly receive the purified product pool comprising the protein from the second chromatography system, thereby obtaining a virus-free filtrate comprising the protein; and

[0391] (h) an ultrafiltration / diafiltration system adapted to fluidly receive the virus-free filtrate from the second chromatography system, or from the third chromatography system and / or the viral filtration system, thereby obtaining a purified protein drug substance;

[0392] Automated equipment controlled by a process automation system (PAS).

[0393] Embodiment 46: The automated installation of embodiment 45, wherein a plurality of reservoirs, each adapted to contain a concentrated medium component solution or an aqueous diluent, are fluidly connected to a perfusion bioreactor directly or indirectly via an optional mixing vessel adapted to receive concentrated medium component solutions and aqueous diluents in a predetermined ratio from the plurality of reservoirs and mix them simultaneously, the optional mixing vessel being fluidly connected directly to the perfusion bioreactor.

[0394] Embodiment 47: The process according to any of embodiments 42 to 44 or the automated installation according to any of embodiments 45 to 46, wherein the protein of interest is a recombinant protein and / or a therapeutic protein.

[0395] Embodiment 48: The automated installation according to any of embodiments 45 to 46, wherein the protein of interest is a recombinant protein and / or a therapeutic protein.

[0396] Embodiment 49: The automated equipment of any of embodiments 22 to 42, any of embodiments 45 to 46, or any of embodiments 48 to 49, wherein the equipment is configured to operate in a continuous manner.

[0397] Embodiment 50: The process of any one of embodiments 1 to 21 or any one of embodiments 42 to 44, wherein the process is carried out in a continuous mode.

[0398] Embodiment 51: The process of any of embodiments 1 to 21 or any of embodiments 42 to 44, wherein the first chromatographic system is disinfected with a chemical disinfectant solution comprising peracetic acid prior to use.

[0399] Embodiment 52: The process of any of embodiment 4 or embodiments 43 to 44, wherein the ultrafiltration / diafiltration system comprises a single-pass tangential flow filtration (SPTFF) and the operating pressure of the SPTFF is controlled in the range of about 0.25 psi to about 60 psi.

[0400] Embodiment 53: The process of any of embodiment 4 or embodiments 43 to 44, wherein the UF / DF system comprises an in-line depth filtration (ILDF) and the operating pressure of the ILDF is controlled in the range of about 0.25 psi to about 60 psi.

[0401] Embodiment 54: A process described in any of embodiments 52 to 53, wherein the operating pressure of the SPTFF and / or ILDF is controlled in a range selected from the group consisting of about 0.25 psi to about 45 psi, about 0.25 psi to about 30 psi, about 0.25 psi to about 15 psi, and about 0.25 psi to about 5 psi.

[0402] The following examples are illustrative and should not be construed in any way as limiting the scope of the invention.

[0403] example Example 1. Demonstration of continuous perfusion culture and protein product capture chromatography for extended production culture periods.

[0404] Materials and Methods

[0405] A series of three engineered runs were performed at 500 L bioreactor scale to demonstrate the process of the invention for producing purified proteins (in this example, recombinant therapeutic protein drug substances) involving the use of co-mixed concentrated media components. A corresponding 2 L satellite bioreactor was operated to generate data using 1× delivery media at smaller scale for comparison.

[0406] Protein of interest, host cells, media. The recombinant therapeutic protein of interest produced and isolated for demonstration purposes was an IgG1κ isotype monoclonal antibody produced by a recombinant CHO-K1 cell line cultured in a chemically defined cell media.

[0407] Perfusion bioreactor and first chromatography system. The perfusion bioreactor was an Xcellerex® XDR 500-L disposable (stirred tank) bioreactor (SUB, GE Healthcare Life Sciences) connected to a Spectrum Krosflo® KPS-600 perfusion system (Repligen). The Xcellerex® XDR 500-L SUB had a blending time of 30-55 seconds with agitation speeds of 95-150 rpm. Shorter blending times are possible by increasing agitation, but these were not characterized. The perfusion system was equipped with a 0.2 μm pore hollow fiber filter that retained cells on the retentate side and allowed high product passage on the permeate side. During the initial start-up of the perfusion culture, the permeate fluid waste was sent directly to the drain via a disposable air break assembly. When the Protein A chromatography product capture run was started, the permeate flow was diverted to the filter cart and the disposable air break assembly was stored in Minncare Sterilant peracetic acid solution (Mar Cor Purification). The DeltaV automated filter cart contained a primary and pre-sterilized grade filter (Express® SHC, 0.2 μm, MilliporeSigma) that acted as a guard filter for the primary capture chromatography column. Other 0.2 μm filters that can be used in the filter cart include Sartopore® 2 (Sartorius), Pall Fluorodyne® EX grade EDF filters, etc. An optional heat exchanger with disposable bag assembly can effectively control the temperature of the chromatography load material but was not used in these runs. However, in other embodiments of the process and automation equipment, disposable heat exchangers are used (Thermo Scientific™ DHX™ Heat Exchanger with Thermo Scientific™ ThermoFlex™ Recirculating Chiller, and Thermo Scientific™ DHX™ Bag Assembly).

[0408] A 200L portable mixer served as a disposable surge vessel (SUSV), which was used as a pressure break between the pumps of the perfusion system and the first chromatography system to manage mismatched flow rates between these two fluidly connected sequential unit operations. The multi-column capture chromatography system used a continuous disposable multi-column chromatography system (Cadence™ BioSMB® PD (hereafter abbreviated as "BioSMB"), Pall Life Sciences), which is a multi-column continuous chromatography system (MCC) designed with a fully disposable flow path, to operate three 14 cm-D x 5 cm-H acrylic columns packed with Protein A resin for this process. The elution outlet of the BioSMB system was connected to two alternating elution pool collection vessels to allow for the simultaneous collection of the elution pool while further processing the low pH virus inactivation and neutralization steps. A schematic partial process flow diagram of the system is shown in Figure 1B. In FIG. 1B, a filter cart is located upstream of the SUSV (labeled "Non-Batch Unit (B1)" or "200L Portable Mixer" in FIG. 1B) and contains a 0.2 μm filter (e.g., Millipore Express SHC, Sartorius Sartopore2, Pall Fluorodyne EDF) to filter the perfusion permeate before it is loaded into the Protein A affinity chromatography column of the first chromatography system, with the filter acting as a guard filter to protect the first chromatography system from particulates. Also shown in FIG. 1B is an optional heat exchanger of disposable plate and frame design that can be used to cool the warm perfusion permeate fluid to room temperature or different desired target temperatures of the SUSV and the first chromatography system.

[0409] Aseptic operation of the process of the present invention was ensured by controlling bioburden using either gamma irradiated disposable components or pre-assembled autoclaved components throughout the connected flow paths. Examples of gamma irradiated components include the Xcellerex® SUB bag, SUB and perfusion system related assemblies, air break assembly (FIG. 2), sterilizing grade filters installed in the filter cart, SUSV mixer bag, elution collection bag, BioSMB manifold, and all associated tote bags for media and buffers. Examples of pre-assembled autoclaved components include hollow fiber filters and valve blocks connected to the chromatography columns to perform the resin disinfection procedure. The entire system boundary was maintained as a fully closed system by using disposable sterile connectors or by functionally closing the system through the use of chemical low-temperature sterilants.

[0410] Operation and Monitoring of the 500-L Disposable Bioreactor (SUB). The control parameters, target set points and acceptable operating ranges for the 500 L SUB culture are listed below in Table 1B.

[0411] [Table 1B]

[0412] The production culture dissolved oxygen (DO) control and pCO2 stripping strategy is shown in Table 2 below. When the air to the T-sparger was increased to 10 SLPM, the overlay was reduced to 0 SLPM. When the offline pCO2 was 152 mmHg, additional air was added to the T-sparger in 5 SLPM increments (total of 10 SLPM or less). The 500L disposable bioreactor (SUB) sparger specifications were as follows:

[0413] T-type sparger: 2mm perforation, agitator base: 2μm sintered disc.

[0414] [Table 2]

[0415] The 500 L (450 L working volume) culture was minimally sampled daily. Viable cell density, culture viability, offline pH, offline pCO2, glucose concentration, lactate concentration and osmolality were measured and recorded. Online agitation, temperature, pH, dissolved oxygen and back pressure were recorded, as were antifoam addition, air and oxygen gassing rates, cell bleed and perfusion rates. Sterility samples were also taken from the bioreactor and perfusion permeate to determine titer. The cell bleed was adjusted to maintain a viable cell density of approximately 50 million viable cells per mL (MVC). This was done using a cell bleed calculator tool developed using Excel™ software (Microsoft Corporation). As the cell bleed rate was adjusted, the perfusion rate was also adjusted to maintain a total outflow rate of 625 mL / min or less when perfused at 2.0 working volumes / day.

[0416] Delivery of media concentrate to the disposable bioreactor (SUB). The sterile perfusion medium was designed to be a concentrated stock solution that was stable at room temperature. To meet these design requirements, in one embodiment, the medium was split into the following three sterile concentrated media component solutions and an aqueous diluent component, each stored in a disposable reservoir:

[0417] (Concentrated Media Component Solution #1) 7.5x (w / w) concentrated media solution (2x-10x is typically useful, but this depends on the media formulation, as the upper limit is determined by the composition of the media and the amount of dry components added),

[0418] (Concentrated Media Component Solution #2) A 20x (w / w) concentrated supplement stock solution containing cystine, tyrosine, and a surfactant (CSSS, a 20x to 100x concentrated supplement stock solution is typically useful);

[0419] (Concentrated Media Component Solution #3) 50% (w / v) glucose, and

[0420] (4) Water for injection (WFI) as the aqueous diluent.

[0421] A schematic partial process flow diagram of the media concentrate delivery strategy at 500 L scale is shown in Figure 1A. The four components listed above were delivered directly to the bioreactor and relied on agitation within the bioreactor to mix the four components. The flow rates of 7.5x medium, 20x CSSS, and 50% glucose concentrate were set manually using calibrated peristaltic pumps and monitored using an in-line Sonotec IL.52 flow meter to ensure accurate delivery. Aqueous diluent (water for injection (WFI)) was delivered to the bioreactor as needed to maintain the bioreactor level set point. The 7.5x medium and 50% glucose solutions were delivered to separate ports at the top of the bioreactor. The WFI and 20x CSSS solutions were tied into separate ports to minimize precipitation of the CSSS solution. According to the present invention, subsurface addition of different concentrated media component solutions and aqueous diluent is preferably avoided. On-demand delivery of all media component solutions and aqueous diluents (e.g., WFI, 7.5x (w / w) concentrated media solution, 20x (w / w) cystine / tyrosine / surfactant (CSSS) stock solution, and 50% (w / v) glucose) through separate ports can also be achieved by using a ratio-controlled pumping skid and automation to maintain the culture volume within the perfusion bioreactor.

[0422] For the demonstration run, perfusion began at 0.5 vessel volumes per day (vvd) on day 2 of production, ramped to 1 vvd on day 4, and 2 vvd on day 6. The inlet flow rates for concentrated media component solutions are shown below in Table 3 along with the estimated flow rate for WFI Trim (average expected inlet flow rate). In Table 3, flow rates are also shown for the permeate flow rate before cell bleed was initiated, and at some exemplary cell bleed rates.

[0423] [Table 3]

[0424] Operation of the first chromatography system and chromatography column disinfection. The first chromatography system was configured to capture the recombinant therapeutic protein of interest into a protein isolate fraction in three exemplary demonstration runs. The first chromatography system included three Protein A affinity capture columns (14 cm diameter x 5 cm height) on a Cadence™ BioSMB PD continuous disposable multi-column chromatography system (also referred to herein as "BioSMB"; Pall Life Sciences). MabSelect™ SuRe™ Protein A affinity matrix on highly cross-linked agarose resin (GE Healthcare Life Sciences) was used for the first two runs, and Amsphere™ A3 Protein A chromatography resin (JSR Life Sciences) was used for the third run. The titer in the permeate was expected to be approximately 0.6 g / L, so it was set at 83 column volumes (CV) with a 50 g / Lr load for MabSelect™ SuRe™ and 108 CV with a 65 g / Lr load for Amsphere™ A3. The method parameters are summarized in Table 4 below. Elution collection used dynamic peak collection based on baseline to baseline absorbance at 280 nm wavelength (peak collection starts at 0.1 absorbance units (AU) and ends at 0.1 AU).

[0425] The BioSMB process was designed to allow the load flow rate to be switched between high, medium, and low flow rates. This switching of flow rates helps manage mismatched flow rates between connected unit operations, i.e., permeate flow rate from the perfusion system and the load flow rate of the BioSMB system. These demonstration runs were operated with the inlet medium component solutions added to the bioreactor at fixed flow rates, while the cell bleed and permeate outlet flow rates were changed daily. The range of potential cell bleed rates is shown in Table 3, which set the range of permeate flow rates to SUSV1 from 531 to 609 mL / min. The BioSMB medium load flow rate changed slightly between demonstration runs, while the high and low flow rates were set to ±10% of the medium flow rate, wider than the expected range of permeate flow rates. A schematic of the SUSV volume control is shown in Table 4 below and in Figure 3, with example flow rates used in one of the demonstration runs. A description of the automation used to switch flow rates is in the next section.

[0426] Prior to the start of the BioSMB capture step for each run, the resin was packed into a glass column housing, and the resin and housing were aseptically chemically sanitized to render the BioSMB flow path functionally closed. Autoclaved valve block assemblies were attached to the inlets and outlets of each column housing, and the columns were attached to the BioSMB manifold, sanitizing solution bags, and trash bags using sterile connectors. A 30 mM peracetic acid (PAA) solution was used as the chemical sanitizer, selected for its effectiveness as a sporicidal agent, yet gentle enough to minimize any damage to resin function. (See, e.g., Jungbauer et al., Method for sterilizing liquid chromatography resins highly resistant to oxidation and a sterilization solution for use in therein, U.S. Patent No. 5,676,837.) A schematic diagram of the column housing chemical sanitization setup of one embodiment is shown in FIG. 4.

[0427] Briefly, the column housings were filled with affinity chromatography resin (open to air). The valve block was autoclaved. Referring to FIG. 4, each column offline on the BioSMB skid was processed in the following manner: A standalone peristaltic pump was used to prime PAA into a disposable bag attached to the vent valve (V4 to V3 in FIG. 4). PAA was flushed with 3 column volumes (CV) through the PAA inlet and outlet valves into a disposable collection bag attached to the sterile connector A (V4 to V2 to V5 to V7 in FIG. 4). PAA was held in each column for ≥15 min, and then the column was flushed again with 3 CV of equilibration buffer (EQ) or storage buffer (V4 to V2 to V5 to V7 in FIG. 4). Each column was then attached to the chromatography skid through the process inlet (V1 in FIG. 4) and process outlet (V6 in FIG. 4) via the sterile connector B connector. After this, the skid line was primed through the vent valve to the disposable bag (V1-V3 in FIG. 4) and the column was prepared for the BioSMB run and functionally closed for this disinfection procedure.

[0428] Alternatively, PAA disinfection of packed chromatography columns can be performed offline as described above, but with flushing of EQ or storage buffer on a simulated moving bed (SMB) skid (washing from V1 to V2 to V5 to V6 in FIG. 4). A disposable bag containing PAA solution can be attached to the column inlet (V2 in FIG. 4), and all disinfection and flushing procedures can be performed on the column on the SMB skid. Disinfectants other than PAA can be used instead, but these must be sporicidal. (See, for example, Jungbauer et al., Method for sanitizing liquid chromatography resins highly resistant to oxidation and a sterilization solution for use therein, U.S. Pat. No. 5,676,837; Monie et al., Sanitization method for affinity chromatography matrices, WO 2016 / 139128 and U.S. Pat. App. Pub. No. 2018 / 036445).

[0429] Chemical disinfection procedures during the demonstration runs were performed only once at the beginning of each run. The Protein A affinity chromatography method itself used a 0.1 M NaOH regeneration wash step, but this disinfectant was not expected to be strong enough to have bactericidal and sporicidal capabilities. In the demonstration runs, the chemical disinfection procedures were performed offline on the BioSMB skid, but the disinfection procedures can be performed on a skid equipped with a BioSMB manifold.

[0430] [Table 4]

[0431] Automation and disposable surge vessel volume control. A process automation system (PAS) was employed to provide flexible process control and management of skid-based and mobile production equipment in support of scalable continuous capture biological production campaigns. Automation also provides autonomous batch reporting, data collection, and material tracking. This was a reusable class-based design and architecture that could be rapidly deployed across production equipment of the same class and configuration. A high-level process automation overview showing communication between equipment types is shown in the schematic diagram of one embodiment of the present invention (Figure 5).

[0432] FIG. 5 shows a schematic diagram of various hardware and software components of an exemplary embodiment of an automated facility for producing purified therapeutic protein drug substance of the present invention that allows communication of data between different components of the system. In particular, FIG. 5 shows several connection interfaces (e.g., Profibus drops) included in the skid of the single-use bioreactor system, the perfusion system, and the continuous first chromatography system. The connection interfaces can provide logical and / or physical connections between the components of the system. In situations where the interface allows for a physical connection, the connection interface can connect to a hardware component such as an Ethernet / Internet Protocol (IP) gateway. One or more dongles can be coupled to portable instruments such as the filter bank, the first mixing tank, and the second mixing tank. The dongles can store and / or communicate information related to the control of the portable instruments to the production facility control system. In certain situations, one or more of the portable instruments can internally store the information stored in the dongle and can function as an internal dongle.

[0433] In the illustrative example of FIG. 5, the various devices can communicate using one or more Profibus communication protocols. In various embodiments, the control of the perfusion system and the continuous first chromatography system can be configured to be set and / or adjusted based on information regarding the operation of at least one of any unit from among the filter bank, the supply tank A, the supply tank B, the collection tank A, and the collection tank B. In the illustrative example of FIG. 5, the collection tank A can have a logically derived software connection with Dongle 1 coupled to the first portable mixing tank. Furthermore, the skid of the continuous chromatography system can be physically connected to the filter bank and the first portable mixing tank through a gateway device. Dongle 2 is coupled to the filter bank and can provide information regarding the operation and identifier of the filter bank. In certain circumstances, data related to the operation of the filter bank can be used to control the continuous chromatography system. Furthermore, Dongle 3 is coupled to the second mixing tank and can provide information regarding the operation and identifier of the second mixing tank. For example, dongle 1 can indicate that a first mixing tank serves as a collection tank for a perfusion system, while dongle 3 can indicate that a second mixing tank serves as a collection tank for a continuous chromatography system.

[0434] While the illustrative example in FIG. 5 shows various software and physical connections between components of the automated equipment of the present invention for manufacturing a purified therapeutic protein drug substance, it is understood that the physical connections may be replaced by software connections, particularly in additional implementations of a purified therapeutic protein drug substance production line, while some of the software connections may be implemented as hardware connections in some additional implementations of a purified therapeutic protein drug substance production line.

[0435] Briefly, the automation of SUSV1 level control relies on the specification of preset volume range limits where control actions are performed. For example, in FIG. 3, when the volume of SUSV1, or any other SUSV in a continuous or semi-continuous process flow, such as SUSV2 or SUSV3 or SUSV4 or SUSV5 ("SUSV" in FIG. 3), reaches a low volume alarm and a high volume alarm, the SMB (or other process skid, such as a virus filtration or UF / DF skid) automatically switches to a low flow rate method and a high flow rate method, respectively. The low and high flow rates of the SMB are selected to be outside the range of expected permeate flow rates entering SUSV1, so that the SUSV1 volume is returned to the center point volume. When the center point volume is reached, the SMB flow rate returns to its intermediate flow rate method. When SUSV1 reaches a low / low ("LL") alarm, the SMB flow rate is stopped, and conversely, when SUSV reaches a high / high ("HH") alarm, the perfusion permeate flow rate is stopped.

[0436] Operation of a 2L bioreactor satellite for comparison with the 500L bioreactor. The bioreactor satellite was performed in a 2L autoclavable glass bioreactor (Applikon) using a BPS-i100 perfusion system (Levitronix). Sterile bags were used to transfer cell culture from the 500L SUB to the 2L bioreactor targeting the inoculum cell density. All concentrated media component solutions and WFI were sourced from the manufacturing facility and reconstituted to 1× formulations. Two different 1× formulations were made at 8 g / L and 12 g / L glucose to accommodate the range of cell densities during production. The satellite was either controlled to the same target set points as the SUB or scaled down accordingly. O2, CO2, and air flows were controlled using a rotameter, and overlay and air sparge flows for CO2 stripping were scaled by vessel volumes per minute (VVM). Agitation was scaled by power per unit volume (P / V). pH was controlled using a ±0.02 dead band. 2 0.02m 2A hollow fiber perfusion filter (0.2 μm pore size) was used in parallel and run 1 was matched with the permeate flux through the filter by recirculating the permeate back to the bioreactor. The permeate recirculation was discarded for subsequent runs for ease of operation. A cell bleed calculator was used to control cell density at both large and small scales. The formula used the current and previous day's offline cell counts to calculate the apparent growth rate and required cell bleed rate required to control at a particular target viable cell density.

[0437] Results and Discussion

[0438] Continuous perfusion culture performance. Cell culture performance results are shown for three 500 L demonstration runs and corresponding 2 L satellite runs, with viable cell density (VCD) shown in Figure 6, viability shown in Figure 7, cell bleed rate shown in Figure 8, and permeate productivity shown in Figure 9.

[0439] Cell density was well controlled to a target of approximately 50 million viable cells / mL (MVC / mL), with a higher bleed rate used at the beginning of the culture and tapered to a lower bleed rate over the duration of the culture. A slightly different cell bleed strategy was used in run 1, which resulted in a more variable growth profile. Subsequent runs transitioned to a cell bleed strategy based on the previous day's growth rate, which resulted in a more tightly controlled VCD. Viability was maintained above 70% for the duration of the cell culture, up to 26 days. Permeate productivity achieved approximately 1 g / L / day for this cell line and process, and the perfusion filter was able to maintain a high product passage for the entire duration of the run (data not shown).

[0440] Media concentrate delivery performance. Multiple performance markers were evaluated to assess the accuracy of media concentrate delivery at the 500 L scale. First, flow verification was performed for the individual media component solutions to ensure that the in-line flow meters were providing accurate readings. Next, Figure 10 shows that the SUB level control operated as intended, with the culture volume in the bioreactor maintained at 450 L and the WFI trim flow rate turned on as needed to maintain culture volume. In addition to these operational checks, the cell culture data shown in Figures 6, 7, 8, and 9 indicate similar performance in terms of cell growth, viability, and productivity between the 500 L scale operated with concentrated media component solutions and the 2 L comparison satellite operated with 1x delivery media.

[0441] Additional metabolic data comparing the 500 L demonstration run 3 with its 2 L comparison satellite is presented: osmolality in Figure 11, CO2 levels in Figure 12, base usage for pH control in Figure 13, lactate ratio production in Figure 14, and specific glucose consumption in Figure 15. All of these trends indicate similar performance between the 500 L scale operated with concentrated media component solutions and the 2 L satellite operated with 1x delivery medium, with particular emphasis on the osmolality profile, which is an indication of media component concentrate addition to the bioreactor and consumption by the cells (see Figure 11).

[0442] Performance of the Continuous Capture Simulated Moving Bed (SMB) First Chromatography System. Simulated moving bed (SMB) first chromatography system BioSMB continuous capture performance results are shown for demonstration run 2, which had the longest duration of operation of the three runs, completing a total of 72 cycles per column (216 total completed elution cycles) over 17 days of continuous operation. Protein A elution ultraviolet (UV) absorbance (A280) profiles are shown as daily snapshots for each column (Figure 16A) along with the elution column volume (CV) for each elution cycle (Figure 16B). Elution peak widths were similar among the three columns, but all columns showed some degree of peak broadening towards the later portion of the run. This could be attributed to an increase in permeate titer, and therefore eluted mass, over the duration of the run, but could also be due to changes in column performance due to aging of the resin. The resin in this run had previously been cycled 60 times, so the total cycles at the end of run 2 were 132. Protein A step yields, shown as the combined daily elution cycle pools in Figure 17, were similar over the course of 72 cycles. Process-related impurities in the combined daily elution pools (neutralized to pH 5 and 0.2 μm filtered) are shown in Figure 18. Impurity levels were relatively consistent between the daily elution pools, indicating consistent performance of the resin over its life.

[0443] The corresponding changes in SUSV level control and BioSMB loading flow rate are shown in Figures 19A-19B. According to the trends of SUSV1 volume and BioSMB loading flow rate, when SUSV1 reached a low volume of 70 L, the BioSMB flow rate shifted to a low flow rate set point, and at a high volume of 130 L, the BioSMB flow rate shifted to a high flow rate set point. After a lower or higher BioSMB flow rate brought the SUSV1 volume back to 100 L, the flow returned to the center point.

[0444] Bioburden Control. Bioburden and endotoxin testing was performed at multiple sample locations over the run period. Results from Demonstration Run 2 are shown in Table 5. For this run, the production bioreactor was run for 25 days and the first chromatography system (here, BioSMB) was run for a total of 17 days. Perfusion was started on day 2, the Protein A column was sanitized on day 7, BioSMB was started on day 8, and the first neutralized Protein A pool was sampled on day 9. Bioreactor samples were whole broth from the bioreactor, PERM samples were taken from the permeate side of the perfusion filter, SUSV1 was taken directly from the vessel, NPRA was taken directly from the daily neutralized elution collection vessel, and 2ndP was taken from the BioSMB outlet for the second pass through flow through. All bioburden results were negative (0 CFU / 10 mL). All endotoxin results were negative except for the 18 second pass through flow through which showed some gel clotting at the lowest dilution. This may have been a sampling issue as the system had clean endotoxin results the day before and after that sample and the corresponding bioburden samples were clean.

[0445] These results confirm that a closed sterile system boundary was maintained from the single-use bioreactor and its associated multiple different concentrated media component solutions and aqueous diluents to the BioSMB and its associated columns, buffers and elution vessels. Additionally, the results validate the use of a single-use air break drain for the permeate and chromatography system waste lines, as well as the sporicidal disinfection of the columns and resins using peracetic acid disinfectant.

[0446] In summary, the above demonstration runs showed consistent performance of the 500L continuous perfusion process, demonstrating the ability to maintain target viable cell density (VCD) for at least 26 days with high viability and high product passage through the perfusion filter. Accurate delivery of media concentrate was demonstrated, i.e., there was comparable performance between the 500L SUB operating with multiple different concentrated media component solutions and aqueous diluents and the 2L comparison satellite bioreactor operating with 1x media delivery. There was consistent performance of the first chromatography system, including the Protein A affinity chromatography capture step, in terms of elution yield and impurity levels. Operation of automated SUSV1 volumetric control with flow rate toggling of the first chromatography system load was demonstrated, as well as a disposable air break drain.

[0447] Use of the process and automated equipment of the present invention allows for the maintenance of a sterile closed system. Practice of the present invention is facilitated by the demonstrated ability to make the system functionally closed and sterile through the use of disposable components, e.g., disposable bioreactors, disposable mixer bags, disposable assemblies, disposable simulated moving bed flow paths, and the use of sporicidal disinfection methods for the chromatography resins and column housings of the chromatography system.

[0448] [Table 5]

[0449] Example 2. Viral inactivation and neutralization system and further downstream processing.

[0450] Following Protein A affinity chromatography via the first chromatography system, downstream unit operations can be performed in batch mode or continuously or semi-continuously.

[0451] In an exemplary embodiment, a two-tank automated virus inactivation system was connected to the elution outlet of the BioSMB system to evaluate a batch low pH virus inactivation step operated with a continuous inlet flow. The elution collection from the BioSMB Protein A affinity chromatography (first chromatography system) was alternated between two 50 L disposable mixer vessels. When the volume in one vessel reached its predetermined value, in this case a target of about 20 L, the acid titration for the low pH virus inactivation process was started in that vessel and the elution collection was switched to the other vessel. For the batch virus inactivation operation, 2 M acetic acid was added stepwise to a target pH of 3.5 and the vessel was mixed with a bottom stirrer and a recirculation pump. The pool was incubated for 60 minutes and then 2 M tris(hydroxymethyl)aminomethane ("Tris") base was added to a target pH of 5.0 in a manner similar to the acid titration. The neutralized virus inactivation product pool was then transferred from the system. A total of six cycles were performed, three times for each tank, with both the low pH and neutral set points achieved within the target pH range of ±0.1. Titration times for acid and base addition were approximately 10 minutes each.

[0452] Figure 21 shows a comparison of high molecular weight (HMW) measured by SE-HPLC between protein isolate fractions after Protein A chromatography and the low pH viral inactivation and neutralization (VI / Neut) viral inactivation product pool. The viral inactivation step was performed manually on day 10 of production, whereas it was performed on days 11-16 with an automated system. The results in Figure 21 show that the high molecular weight (HMW) between protein isolate fractions after Protein A chromatography and the viral inactivation product pool after VI / Neut is comparable, indicating that the acid and base titration procedure of the viral inactivation system did not affect the product quality.

[0453] The following is another non-limiting example of a downstream batch mode operation, where all steps use disposable (single-use) components.

[0454] Several days of BioSMB (first chromatography system) run including Protein A affinity chromatography are pooled into protein isolate fractions for viral inactivation (VI). Viral inactivation is performed by lowering the pool to pH 3.5 with 2M acetic acid. The acidified pool is held for 1 hour. After the low pH hold, the viral inactivation product pool is neutralized to pH 5.0 with 2M Tris base. The neutralized viral inactivation product pool is filtered through a Millistak+® HC Pod A1HC depth filter (MilliporeSigma) followed by a sterilizing grade filter to clarify the viral inactivation product pool before introducing it into the second chromatography system.

[0455] The second chromatography system includes a cation exchange (CEX) column in flow-through mode. This step utilizes Fractogel® EMD(M) COO-resin (MilliporeSigma). CEX is followed by further chromatographic purification via a third chromatography system, such as but not limited to mixed-mode chromatography (MM) using Capto™ Adhere (GE Healthcare Life Sciences) resin. Loading adjustments of pH and / or conductivity can be performed prior to loading (in-line or batch) onto the MM column. This step is performed in flow-through mode.

[0456] Viral filtration (VF) is performed on the MM flow-through pool as an orthogonal method to remove viral particles by size to obtain a virus-free filtrate containing the recombinant therapeutic protein. Viral filtration can be performed using, for example, but not limited to, Planova™ 20N filters (Asahi Kasei). The virus-free filtrate pool is concentrated and diafiltered using, for example, but not limited to, 30 kD regenerated cellulose filters (MilliporeSigma) to produce the purified therapeutic protein drug substance in the formulation buffer at the target product concentration.

[0457] Example 3. Peracetic acid (PAA) disinfection of Protein A matrix.

[0458] The success of continuous processing can be facilitated by extending the sterile envelope from the perfusion bioreactor to the Protein A affinity chromatography capture step (or first chromatography system) and to downstream processes by the use of sterile disposable components and equipment. However, it is difficult to guarantee the sterility of in-house packed chromatography columns, and gamma irradiated columns are expensive and not widely commercially available. To address the need for sterile chromatography columns, we have developed a chemical column disinfection process that allows the capture step to be performed continuously in a sterile manner, assuming the use of sterile connectors, while thoroughly inspecting the integrity of any welds in the system.

[0459] Materials and Methods. The materials listed in Table 6 were used in the filling and disinfection procedures described in this Example 3. Rehydrated inocula of the individual bacterial species listed in Table 6 were prepared from BioBall® Multishot 10E8 kits (bioMerieux SA) according to the manufacturer's instructions, with an average of 0.7-1.5x10 8 cfu with a standard deviation less than 20% of the mean. Rehydrate into 1.1 mL of BioBall® Rehydration Fluid and add 10 x 100 μL doses of 10 7 cfu was obtained.

[0460] [Table 6]

[0461] Primary column packing and sanitization procedure. Ethanol (70% v / v) was added to the empty column housing. Air was removed from under the bottom frit by aspirating it through the column outlet with a peristaltic pump and manually directing the air bubbles with a small paddle. Once all air was removed, the appropriate amount of MabSelect® SuRe Protein A resin to pack either a 5 cm or 10 cm resin bed was added to a BPG 140 / 500 glass chromatography column housing. A peristaltic pump was used to remove the storage buffer through the outlet of the column. The storage buffers shown in Table 6 are only examples, alternatively the storage buffer may be any buffer the resin was shipped in, for example a buffer containing 20% ​​ethanol, or EQ or other buffer or deionized water if settling / decanting the resin was previously used to remove particulates or to obtain an accurate slurry percentage.

[0462] Approximately 3 column volumes (CV, based on settled bed height) of either Milli-Q® purified water or Protein A equilibration buffer (EQ) were added to the top of the settled bed. This volume was drawn through the settled bed using a peristaltic pump to remove residual storage buffer. Approximately 2 CV of Milli-Q® purified water or EQ buffer was added to the settled bed and the bed was gently slurried with a small paddle. To ensure the level of bioburden and demonstrate the effectiveness of the disinfection procedure, B. subtilis or a cocktail of organisms (see Table 6) was added to the slurry at approximately 100 CFU / mL of slurry volume. The bed was allowed to settle using a peristaltic pump to remove excess.

[0463] Approximately 3 CV of 0.7% (v / v) PAA was added on top of the settled bed and this volume was pumped through the settled bed using a peristaltic pump to remove residual water (or Protein A EQ). Peracetic acid (0.7% v / v) was added to the column to create an approximately 50% slurry of resin and PAA. The resin was slurried with a paddle. The resin slurry was allowed to settle long enough to create a liquid layer on top of the bed large enough to cover the column top adapter up to the adapter O-ring. The top column adapter was put in place and lowered until the O-ring was covered by the PAA solution. The top adapter was manipulated to remove residual air from the frit and O-ring. The top adapter was immersed in the 0.7% PAA solution for approximately 20 minutes and then the O-ring was tightened. The top adapter was lowered into the PAA solution to push the PAA solution up into the center column tube. The center column tube was then connected to the 0.7% PAA loading solution. The column was packed with 0.7% PAA solution at 380-550 cm / hr, although higher packing rates, e.g., 600 cm / hr, are possible.

[0464] The following procedure was used to mimic the steps a packed column undergoes in a Good Manufacturing Practice (GMP) production run. All steps were performed at a flow rate of approximately 150 cm / hr. 10 CV of deionized water was run through the column to remove the PAA solution. 3 CV of sterile 0.1 M NaCl was run through the column to simulate a Height Equivalent Theoretical Plate (HETP) test. 3 CV of storage buffer was run through the column and the column was stored overnight before being sanitized with PAA.

[0465] The column was sanitized with 0.2% (v / v) PAA. 5 CV of 0.2% PAA was run downflow through the column and 5 CV of 0.2% (v / v) PAA was run upflow through the column. The column was held in 0.2% PAA for 60 min, after which Protein A EQ buffer was run through the column to remove the PAA solution. Samples were taken at 5 CV and 7 CV and submitted for bioburden analysis. The column was then connected to a 2 L bioreactor containing BAK medium. Media was recirculated through the column at approximately 50 mL / min using a peristaltic pump. The column inlet pressure was monitored with a SciLog® (Parker Hannifin) pressure transducer. The pump was set to turn off when a maximum pressure differential of 20 pounds per square inch differential (psid) was reached. The column was left connected to the reactor for 10-14 days. A sample after recirculation was removed from the column at the completion of the experiment and submitted for bioburden analysis.

[0466] Alternative Filling and Sanitizing Procedure 1. An alternative filling and sanitizing procedure was also used following the primary filling and sanitizing procedure above, except that 0.7% (v / v) PAA was used for slurrying, filling, and sanitizing.

[0467] Alternative Packing and Sanitizing Procedure 2. A second alternative packing and sanitizing procedure was also attempted using 0.2% (v / v) PAA with 0.1 M NaCl for slurrying and packing the column. Similar to the primary packing and sanitizing procedure above, the column was sanitized with 0.2% PAA after packing and sodium chloride was added to improve packing performance. However, sodium chloride interacted with the PAA solution and generated air in the column. Therefore, adding sodium chloride together with PAA is not recommended.

[0468] Alternative Loading and Sanitizing Procedure 3. In the third alternative loading and sanitizing procedure, the resin was slurried and loaded in 0.1 M NaCl without PAA. During the first slurry step prior to loading, the bioburden cocktail (see Table 6) was added to the resin to approximately 120 CFU / mL. The 0.1 M NaCl was removed by running approximately 3 CV of water (or ProA EQ) through the column, and the column was then sanitized with 0.2% (v / v) PAA (5 CV down, 5 CV up, 60 min hold), flushed with Protein A EQ buffer, and connected to a 2 L bioreactor. As with previous experiments using the primary sanitizing procedure (above), flush and post-bioreactor recirculation samples were removed from the column at the completion of the experiment and subjected to bioburden analysis. For bioburden testing, approximately 100-200 mL of sample was placed into a sterile sample bag and the bioburden was analyzed as described below.

[0469] Process scale validation of the loading and sanitization procedure. Two 500L engineered runs utilizing BioSMB for continuous capture of the immunoglobulin of interest were performed at a manufacturing plant. The production process used four MabSelect™ SuRe™ Protein A affinity matrix columns, 14 cm diameter x 10 cm bed height, for capture of the bioreactor permeate. The first run used a primary loading and sanitization procedure in which the resin was slurried with 0.7% (v / v) PAA and the column was loaded with 0.7% (v / v) PAA, followed by a 0.2% (v / v) PAA sanitization step. The column was not reloaded for the second run. The second run only used a 0.2% PAA sanitization step before putting the column into operation.

[0470] Bioburden Analysis. Bioburden testing is based on the USP <61> Briefly, 10 mL of sample was aseptically removed from a sample bag containing 100 mL of total collected sample and added to at least 90 mL of sterile phosphate buffered saline (PBS) or sterile water (or a volume of sample and diluent such that the product was not diluted more than 1:10). The total aliquot volume was poured into a Milliflex® filtration system (MilliporeSigma), filtered, and then incubated on Milliflex® agar plates at 30-35°C for tryptic soy agar (TSA) and 20-25°C for Sabouraud dextrose agar (SabDex; SDA) for at least 3 and 5 days, respectively.

[0471] Results Table 7 (below) contains the results for each of the test conditions used in the column disinfection experiments. All conditions tested resulted in a fully disinfected column. No bioburden was found in any of the flush samples, and all conditions resulted in at least 10 days of sterile operation when connected to the 2L bioreactor. The minimum time the column was connected to the 2L bioreactor was 10 days. The 10 day incubation period was chosen because it allows for detectable growth of even slower microbial growth on the chromatography column and in the bioreactor.

[0472] The 0.7% (v / v) PAA slurry and packing procedure (i.e., the primary column packing and sanitizing procedure described above) was considered the most stringent option for disinfection at process scale and was selected for use in engineered operations. Because 0.2% PAA has been shown to be effective against spore-forming bacteria and all resin and column parts were in contact with 0.7% PAA during the packing process, a post-column performance test of the 0.2% (v / v) PAA disinfection step was performed after connecting the column to the BioSMB valve block. A full evaluation of the impact of 0.7% PAA exposure on Protein A longevity had not been performed at the time of these experiments, so a lower concentration was also selected to limit unpredictable results due to the high concentration of PAA disinfectant.

[0473] Table 8 (below) contains the bioburden results from the Protein A affinity chromatography step of the first engineered run. All samples tested were negative for bioburden as described above, indicating that the 0.7% (v / v) slurry / fill procedure was effective in eliminating bioburden and enabled sterile downstream processing for 14 days of continuous downstream processing.

[0474] Table 9 (below) contains the bioburden results from the Protein A affinity chromatography step of the second engineered run. All samples tested were negative for bioburden, and the system was successful in continuous downstream processing for 14 days. However, it should be noted that these columns may have remained sterile from a previous process.

[0475] The 0.7% (v / v) PAA resin slurry and packing procedure followed by 0.2% (v / v) PAA sanitization used in the two engineered runs effectively sanitized the column and enabled continuous Protein A affinity chromatography capture of protein product over a 14 day period. No bioburden was detected in either the Protein A step or the downstream process for the engineered runs (see Tables 8 and 9 below), and the BioSMB skid performed as designed over a 14 day period.

[0476] [Table 7]

[0477] [Table 8]

[0478] [Table 9]

[0479] Example 4. Continuous process from disposable bioreactor (SUB) to final tangential flow filtration (TFF)

[0480] A set of two runs was performed at 500 L perfusion bioreactor scale to demonstrate a continuous embodiment of the process of the invention for producing a purified protein of interest or purified protein drug substance containing the protein of interest from a single-use perfusion bioreactor to a final formulation step. An exemplary process flow diagram is shown in Figure 22. Downstream of the perfusion bioreactor, the following steps were fluidly connected and operated in continuous mode: Protein A affinity chromatography capture step on a Cadence™ BioSMB® PD system (Pall Corporation), low pH viral inactivation (VI) step on a two-tank Pall Cadence™ viral inactivation system, depth filtration step, ion exchange polishing step, and a continuous final formulation step consisting of a two-stage single-pass tangential flow filtration (SPTFF) and in-line diafiltration (ILDF) module. Given the additional cost of the filter and the fact that the resulting material was not for non-clinical or clinical studies, viral filtration could be included in the production of a protein of interest intended for clinical use. Disposable surge vessels (SUSVs) were used between each unit operation to manage flow mismatches in the process and accommodate process upsets. Additionally, the surge vessel before the IEX step was titrated to the pH setpoint with base and the surge vessel after the IEX step was titrated to the pH setpoint with acid. Results are presented for the overall continuous process performance, but focus on the depth filtration and IEX steps as newly added steps to the continuous train. The SPTFF-ILDF process and results are discussed in Example 5 below.

[0481] Materials and Methods. A 500 L disposable perfusion bioreactor was operated in a similar manner as described in Example 1 herein above. The downstream process was operated continuously for 14 days. The first chromatography system used was Protein A affinity chromatography, which was performed on a Cadence™ BioSMB® PD System (Pall; "BioSMB") continuous chromatography system, using four columns. The columns were packed by slurrying the resin with 0.7% (v / v) PAA and packing the resin with 0.7% (v / v) PAA. Packing performance was tested by performing height equivalent to theoretical plate (HETP) and asymmetric analysis. As the packing procedure is a non-sterile step, the columns were sanitized with 0.2% (v / v) PAA after installation in the BioSMB immediately prior to starting the process (see Example 3 herein). The Protein A affinity chromatography step was operated in a similar manner to that described in Example 1, including the use of a surge vessel (SUSV1) between the bioreactor and the Protein A system.

[0482] Low pH viral inactivation (VI) was performed using a Cadence™ viral inactivation system (Pall) that includes two disposable mixing tanks for collection, acidification, and neutralization. The viral inactivation step was operated in a similar manner to the method described in Example 2 hereinabove. Multiple Protein A eluates were collected in one of the two VI disposable mixing tanks. The Protein A elution pool was adjusted to low pH with acid and maintained at this pH for the target incubation time to inactivate any viruses that may be present. After viral inactivation, the pool was adjusted to neutral pH with base. This neutralized viral inactivation product pool (NVIP) was then pumped from the mixing tank to SUSV2, which was located in front of the depth filter cart. During acidification, retention, and neutralization, the next series of Protein A eluates were collected in the second VI disposable mixing tank of the viral inactivation system. The cycle of alternating collection tanks was repeated for the duration of the Protein A process.

[0483] The neutralized viral inactivation product pool (NVIP) was filtered through a depth filter and a 0.22 μm filter using a depth filtration cart (see FIG. 22). A more detailed schematic of the depth filtration cart between SUSV2 and SUSV3 shown in FIG. 22 is shown in FIG. 23. Prior to use, the depth filter was autoclaved at 123.1° C. for 60 minutes to reduce potential bioburden. The depth filter was then placed in a filter holder, flushed, sanitized with 0.5 N NaOH, and equilibrated with buffer. The filter holder was then placed in a depth filtration cart. Two depth filter / final filter assemblies can be attached to the cart. The NVIP material was filtered through one side (DF-1) until the maximum depth filter throughput was reached. At this point, a second depth filter / final filter assembly was brought online to receive the load material. After the load target was reached, the first depth filter assembly was flushed with buffer to collect the residual product from the depth filter. This was done in-line simultaneously with the process by a second pump connected to the depth filter assembly and buffer bag. Once the flush was complete, a new depth filter assembly was installed. Each set of depth filters was autoclaved and cleaned before being installed in the system. This process was repeated at throughput limit until the Protein A cycle was completed.

[0484] As shown in Figure 22, the filtered neutralized viral inactivation product pool (FNVIP) was collected in SUSV3 to an appropriate volume before loading into the second chromatography system, an ion exchange (IEX) column. The IEX flow-through step was performed on an AKTA™ Ready (GE Healthcare Life Sciences) disposable chromatography system. The column and resin were sanitized with 0.5 N NaOH before use. Before the start of the IEX chromatography step, the viral inactivation product pool was pH adjusted by continuously adding titrant to SUSV3 as needed. The absorbance of the IEX column eluate was monitored online at a wavelength of 280 nm and used to collect the IEX pool, and this purified product pool containing the protein was pH adjusted by continuously adding a titrant in SUSV4 for continuous processing, if necessary, and filtered (0.22 μm) to obtain a filtrate before the final UF / DF, which was virus-free in an embodiment that includes a virus filtration system to obtain, for example, a clinically usable protein drug substance. (See FIG. 22.) For this continuous IEX step, a single column was used. Since the unloading step (equilibration, washing, stripping, regeneration) requires the column to be taken out of the load, this resulted in an increase in the SUSV3 volume during the unloading phase of the step. To maintain level control of SUSV3, a flow rate higher than the incoming surge vessel volumetric flow rate was used to reduce the surge vessel volume when the loading phase was resumed. Level control was also maintained in SUSV3 by using automation to vary the pump speed of the IEX chromatography system to maintain a preset volume range in the disposable surge vessel.

[0485] As described herein, the process of the present invention utilizes SUSVs between consecutive unit operations to manage flow differentials, provide pressure breaks between unit operations, and provide time to react to modulations in the system. The automated control strategy of these surge vessels was operated as described in Example 1. Two runs, Run #1 and Run #2, were performed with the upstream unit operations run similarly. For downstream processing, Run #1 differed slightly from Run #2 in that the unit operations in Run #1 were run connected continuously through the IEX step. In Run #1, the SPTFF / ILDF was not connected to the upstream unit operations and was run separately in a semi-continuous fashion. In Run #2, all unit operations were linked and run in a continuous fashion from the perfusion bioreactor to the SPTFF / ILDF step to obtain a purified protein drug substance containing the protein of interest. Some control aspects (such as the level control of SUSV1) were also not operational in Run #1, but otherwise the process steps and operating parameters were configured in a similar manner.

[0486] Results The perfusion culture process was operated for 22-23 days of production. A continuous downstream process was connected to a 500L perfusion bioreactor and operated for a total of 14 days. Results for Run #1 and Run #2 were similar. The overall summary for Run #2 was 39 Protein A cycles per column (156 total elution peaks), 53 VI cycles alternating between two tanks, 4 depth filter cycles (with a switch to a new filter between cycles), and 70 IEX cycles.

[0487] All upstream and downstream steps were operated as fully / functionally closed systems. A "fully closed" system is defined as a process system that does not expose the product to the room environment, and adding materials to the closed system avoids exposure of the product to the room environment. For example, the upstream bioreactor is a fully closed system and is never open to the environment. A "functionally closed" system is defined as a process system that may be opened (e.g., to install a filter or column) but is returned to a closed state by disinfecting the system before product introduction, for example, a downstream system is functionally closed by closing with the use of a disinfectant. (Palberg et al.,Challenging the Cleanroom Paradigm for Biopharmaceutical Manufacturing of Bulk Drug, Substances, BioPharm International Volume 24, Issue 8 (2011)) All systems were constructed with gamma irradiated and autoclaved components, sterile connectors or weldable tubing for connections, and disposable air break assemblies for waste lines to establish closed system boundaries for each step. The system was functionally closed by disinfecting the components that could not be gamma irradiated or autoclaved. The Protein A column and resin were disinfected with peracetic acid (PAA) and the depth filter membrane, IEX column and resin, and SPTFF-ILDF membrane were disinfected with 0.5N NaOH as described in the Methods section of this Example 4. The bioburden and endotoxin data sampled from multiple time points of the process on different days are summarized in Tables 10 and 11 below. The results demonstrate that a state of low bioburden control can be successfully achieved by using appropriate procedures to operate a continuous process as a fully / functionally closed system.

[0488] [Table 10]

[0489] [Table 11]

[0490] Example 5. Single-pass tangential flow filtration / in-line diafiltration (SPTFF / ILDF)

[0491] In this example, a set of two runs of the process of the invention described in Example 4 was carried out in a fully continuous mode from the bioreactor including single pass tangential flow filtration (SPTFF) and in-line diafiltration (ILDF) to the final formulation step. Advantages of SPTFF and ILDF include minimizing the need for large in-process holding vessels or tanks, and reducing the time the product pool needs to be held at conditions that may be less stable than the final formulation conditions. Also, potentially less filter area can be used than traditional batch processes. However, there are challenges to implementing a continuous process format, including long operation times for both the SPTFF and ILDF equipment, and previously unknown fouling characteristics over time, adjustments made by automation to maintain surge tank volumes, and previously unknown effects of different inlet feed flow rates due to flow rate matching between different unit operations, and maintaining clean processing over time. As further described below, the present invention has solved all these challenges.

[0492] Methods and Materials. The preceding manufacturing process steps and disinfection before the SPTFF / ILDF steps are described in Examples 3 and 4 above. For the downstream single-pass tangential flow filtration (SPTFF) and in-line diafiltration unit operations, Cadence™ Single-Pass TFF Module (SPTFF, Pall) and Cadence™ In-Line Diafiltration (ILDF, Pall) devices were used in both runs. Both devices were multi-stage tangential flow filtration (TFF) cassettes, and new membranes were used for each run. The SPTFF and ILDF devices and flow paths are shown diagrammatically in FIG. 24. In the setup for Run #1, all tubing assemblies were autoclaved for the SPTFF portion of the system, but for the ILDF portion of the system, some parts of the system were only disinfected with 0.5N NaOH and not autoclaved, which resulted in bioburden being observed in the ILDF pool. To mitigate this issue in Run #2, all lines, flow meters, and pressure sensors were autoclaved, except for the conductivity sensor, which is not autoclavable. The conductivity sensor was sprayed with disinfectant (Minncare®) and attached to the line in a sterile hood. After the process lines were attached to the SPTFF and ILDF apparatus, the membrane and flow path were disinfected before use with 0.5 N NaOH. Flushing before use was performed at a faster flow rate while keeping the pressure below 20 psi. After disinfection, the system was operated as functionally closed, with all connections made by weldable tubing or commercial AseptiQuik® connections (Colder Products Company). The feed flow rate of the SPTFF (from SUSV4) matched the product effluent flow rate from the preceding column step. The retentate pump controlled the retentate flow rate and thus the product concentration factor in the SPTFF apparatus. In the SPTFF step, the product is concentrated in the filter. The set retentate flow rate (e.g., 5-10 times lower than the feed flow rate depending on the desired concentration factor) determines the amount of buffer removed through the permeate, concentrating the retained product. The ILDF feed was connected to the retentate of the SPTFF with an optional break tank, or surge vessel, between steps.In this example, in run #1, an autoclaved 2 L bioreactor with a mixer served as an optional surge vessel between the SPTFF and ILDF steps, although another type of surge vessel could be used, e.g., a disposable surge vessel (SUSV). The schematic of the SPTFF / ILDF setup (Figure 24) shows the run #1 setup where an autoclaved 2 L surge vessel with a mixer between the SPTFF and ILDF (called the "break tank" in Figure 24) was used for the majority of the run. As shown in the data below, the surge vessel was removed for the last few days of the run. In run #2, no surge vessel was placed between the SPTFF and ILDF unit operations. Run #2 had the SPTFF retentate line connected directly to the ILDF feed line. This connection not only eliminated the need for a surge vessel, but also eliminated pump 2 from the setup. The feed flow rate of the ILDF was matched to the retentate flow rate of the SPTFF. The retentate flow rate of the ILDF was controlled at the same rate as the feed flow rate. A separate diafiltration pump delivered formulation buffer to multiple channels of the ILDF device. Conversion to formulation buffer was controlled by the ratio of the diafiltration feed stream to the ILDF feed / retentate stream. The permeate of both devices was discharged through an air break as described in the previous examples.

[0493] Results The SPTFF and ILDF modules ran consistently in full continuous mode over the duration of both runs with no obvious signs of fouling or performance degradation over the 12 day period as evidenced by consistent pressures of less than 5 psi for SPTFF and less than 15 psi for ILDF throughout the duration of the runs. Both systems recovered quickly after interruptions which shut off all pumps. The product concentration factor was maintained throughout the duration of the runs at a pre-determined value approximately 5-10 times lower than the feed flow rate based on the desired concentration factor and the final conductivity of the pool matched the starting diafiltration buffer. A total of 4.8 kg of product mass output was processed in run #1 and 7.3 kg of product mass output was processed in run #2. The overall yield for run #1 was 98% and the overall yield for run #2 was not calculated.

[0494] The above are merely illustrative, and one of ordinary skill in the art of the present invention can readily modify the ingredients and process parameters as needed for a particular recombinant therapeutic protein drug substance of interest. The present invention includes the following preferred embodiments. (1) A process for producing a purified protein of interest, comprising the steps of: 1. A process comprising: (a) culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that permit the cells to secrete said protein into said liquid medium over a production culture period of at least 10 days, wherein during said production culture period, periodically or continuously, fresh sterile liquid medium that is simultaneously mixed from a plurality of different concentrated medium component solutions and an aqueous diluent is added to the one or more disposable perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors in direct relation to a volume of culture that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed, and wherein the volume of permeate removed is automatically and fluidly fed from the one or more disposable perfusion bioreactors to a disposable surge vessel and then to a first chromatography system, whereby said protein is collected in a protein isolate fraction. (2) The following steps: (b) switching the protein isolate fraction to a low pH or detergent viral inactivation system, and optionally a neutralization system, to obtain a viral inactivation product pool comprising the protein. (3) The following steps: (c) introducing the viral inactivation product pool into a second chromatography system to obtain a purified product pool comprising the protein; (d) switching the purified product pool containing the protein to an optional third chromatography system and / or a viral filtration system to obtain a virus-free filtrate containing the protein; The process of (2), further comprising: (e) switching the virus-free filtrate through an ultrafiltration / diafiltration system to obtain a composition comprising a purified protein of interest. (4) 2. The process according to (1), wherein the protein of interest is a recombinant protein. (5) 2. The process according to (1), wherein the protein of interest is a therapeutic protein. (6) The process of (1), (2), or (3), wherein one or more of the first chromatography system, the second chromatography system, the third chromatography system, the low pH or detergent viral inactivation system, the neutralization system, the viral filtration system, or the ultrafiltration / diafiltration system include one or more disposable components. (7) 2. The process of claim 1, wherein the mammalian cells are cultured in 2, 3, 4, 5 or 6 disposable perfusion bioreactors. (8) The process of (1), wherein the one or more disposable bioreactors can contain a volume of liquid medium of about 50 L to about 4000 L. (9) 2. The process of claim 1, wherein the fresh sterile liquid medium is added to the one or more perfusion bioreactors by directly injecting the different concentrated medium component solutions in a fixed ratio relative to one another into the perfusion bioreactor, while an aqueous diluent is also added in varying ratios to the different concentrated component solutions to maintain a constant culture volume in each perfusion bioreactor. (10) 2. The process of claim 1, wherein fresh sterile liquid medium is added to the one or more perfusion bioreactors by injecting the different concentrated medium component solutions and the aqueous diluent in a fixed ratio relative to one another directly into the perfusion bioreactor to maintain a constant culture volume in each perfusion bioreactor. (11) 2. The process of claim 1, wherein fresh sterile liquid medium is added to the one or more perfusion bioreactors by injecting the plurality of different concentrated medium component solutions and the aqueous diluent in a fixed ratio relative to one another into a mixing chamber to maintain a constant culture volume, where fresh sterile liquid medium is mixed simultaneously before being added to each perfusion bioreactor. (12) The process of claim 1, further comprising: measuring a fluid volume in the disposable surge receptacle using an automatic control device including a detector; and a processor varying a pump speed of the first chromatography system to maintain a preset volume range in the disposable surge receptacle. (13) The process of (2) or (3), wherein one or more of steps (b), (c), (d), or (e) are performed automatically and fluidly with uninterrupted flow from a previous step, a surge receptacle is used during one or more steps, and the processor varies the pump speed in a subsequent step to adjust a preset volume range of the surge receptacle preceding the subsequent step. (14) 14. The process of claim 13, wherein an in-line or in-vessel adjustment of pH and / or conductivity loading is performed between one or more of steps (b), (c), (d), or (e). (15) (i) a process automation system in electronic communication with at least the one or more disposable perfusion bioreactors, the disposable surge vessel, and the first chromatography system; (ii) the process automation system stores a first set of control modules for controlling operation of at least one single-use perfusion bioreactor of the one or more single-use perfusion bioreactors; (iii) the process automation system storing a second set of control modules for controlling operation of the supply tank; (iv) the process automation system stores a third set of control modules for controlling operation of the collection tank; (v) The process of (1), wherein the at least one disposable perfusion bioreactor is logically configured to be coupled to one or more feed tanks, one or more harvest tanks, or a filter bank. (16) 16. The process of claim 15, wherein the at least one disposable perfusion bioreactor is disposed on a skid, the skid including a plurality of communication interfaces for electronically coupling the at least one disposable perfusion bioreactor to a plurality of portable instruments. (17) determining, by the process automation system, that the disposable surge receptacle is coupled to one of the plurality of communication interfaces based on data received via the communication interfaces, the data indicating an identifier of the disposable surge receptacle and a function of the disposable surge receptacle; The process of claim 16, further comprising: determining, based at least in part on the identifier and the functionality of the disposable surge receptacle, that the disposable surge receptacle is a collection tank and that a third set of control modules is for controlling operation of the disposable surge receptacle. (18) determining, by the process automation system, that a mixing vessel is coupled to an additional communication interface of the plurality of communication interfaces based on additional data received via an additional communication interface, the additional data indicating an additional identifier of the mixing vessel and an additional function of the mixing vessel; 18. The process of claim 17, further comprising: determining, based at least in part on the additional identifier and the additional function of the mixing vessel, that the mixing vessel is a supply tank and that the second set of control modules is for controlling operation of the mixing vessel. (19) The process described in (1), wherein the production culture period is at least 20 days. (20) 1. An automated facility for producing a purified protein of interest, said facility comprising: (a) one or more disposable perfusion bioreactors capable of containing a liquid medium under conditions that permit cultured cells to secrete said protein into the liquid medium for a production culture period of at least 10 days, the disposable perfusion bioreactors being adapted to receive fresh sterile liquid medium through a fluid inlet into each of said perfusion bioreactors in direct relation to a volume of conditioned medium that is continuously or periodically removed from each of said perfusion bioreactors as a volume of permeate or cell bleed during said production culture period; (b) a plurality of reservoirs, each adapted to contain a concentrated media component solution or an aqueous diluent, fluidly connected to an inlet to each of the perfusion bioreactors either directly or indirectly through an optional mixing vessel adapted to receive the concentrated media component solution and the aqueous diluent in a predetermined ratio from the plurality of reservoirs and mix them simultaneously, the optional mixing vessel being fluidly connected directly to the inlet to each of the perfusion bioreactors; (c) a first disposable surge vessel (SUSV1) into which the removed permeate volume is automatically and fluidly delivered from the one or more disposable perfusion bioreactors; (d) a first chromatography system adapted to automatically and fluidly receive a cell-free permeate from said SUSV1, whereby said protein is captured in a protein isolate fraction; The automated equipment is controlled by a process automation system (PAS). (twenty one) (e) a low pH or detergent viral inactivation system, and optionally a neutralization system, adapted to automatically and fluidly receive the protein isolate fraction from the first chromatography system, thereby obtaining a viral inactivation product pool comprising the protein; (f) a holding vessel or a second disposable surge vessel adapted to receive the viral inactivation product pool. (twenty two) (g) a second chromatography system adapted to fluidly receive the viral inactivation product pool from the holding vessel or the second disposable surge vessel, thereby obtaining a purified product pool comprising the protein; and (h) an optional third chromatography system and / or virus filtration system adapted to fluidly receive the purified product pool comprising the protein from the second chromatography system, thereby obtaining a virus-free filtrate comprising the protein; and (i) an UF / DF system adapted to fluidly receive the virus-free filtrate from the second chromatography system, or from the third chromatography system and / or the virus filtration system, thereby obtaining the purified protein of interest. (twenty three) The automated equipment according to (20), wherein the one or more disposable perfusion bioreactors can contain a liquid medium having a volume of about 50 L to about 4000 L. (twenty four) The automated equipment of (20) further includes an automatic control device including a detector for measuring the fluid volume of SUSV1 and a processor for varying a pump speed of the first chromatography system to maintain a preset volume range of SUSV1. (twenty five) 21. The automated equipment of claim 20, wherein one or more of the first chromatography system, the second chromatography system, the third chromatography system, the low pH or detergent viral inactivation system, the neutralization system, the viral filtration system, or the ultrafiltration / diafiltration system include disposable components. (26) directly fluidly connected downstream of the first chromatographic system; (i) A second disposable surge container; or (ii) at least two automatically switchable alternative disposable collection vessels (SUCV1 and SUCV2) adapted to receive said protein isolate fractions; 21. The automated equipment of claim 20, wherein (i) and (ii) are adapted to receive the protein isolate fraction from the first chromatography system and fluidly deliver the protein isolate fraction to the low pH or detergent viral inactivation system. (27) The low pH or detergent viral inactivation system, and optionally the neutralization system, (i) a second disposable surge vessel adapted to receive the protein isolate fraction; or (ii) at least two automatically switchable alternative disposable collection vessels (SUCV1 and SUCV2) adapted to receive said protein isolate fractions; 22. The automated equipment according to claim 21, wherein viral inactivation, and if necessary neutralization, is carried out in a second disposable surge vessel, or in SUCV1 and SUCV2. (28) The automated equipment of (20) further comprising a hollow fiber membrane, a series of depth filters, or a filtration cart before the permeate is automatically and fluidly supplied to the SUSV1. (29) 22. The automated equipment of claim 21, further comprising a disposable surge vessel adapted to receive the viral inactivation product pool in (f). (30) The automated equipment of (20) further comprising a heat exchanger upstream of the SUSV1. (31) The automated equipment of (20), further comprising a filtration system upstream of the SUSV1. (32) (i) the second chromatography system; (ii) said optional third chromatographic system; (iii) the virus filtration system, and (iv) the UF / DF system, 23. The automated installation of claim 22, wherein the installation is automatically and fluidly connected to a preceding system, and a surge receptacle is optionally used to regulate the uninterrupted flow of material between the connected systems. (33) the at least one or more disposable perfusion bioreactors, SUSV1, the first chromatography system, the low pH or detergent viral inactivation system, the holding vessel or disposable surge vessel, the second chromatography system, the optional third chromatography system and / or viral filtration system, and the UF / DF system comprise a first device arranged in a first configuration of a production line for the purified protein of interest; The automated equipment of any one of (20) to (22), wherein a first plurality of control modules are implemented to control the operation of the first equipment. (34) a second piece of equipment is disposed in a second configuration of a production line for additional purified proteins of interest, the second configuration of the production line including at least one or more disposable perfusion bioreactors, a first chromatography system, a low pH or detergent viral inactivation system, a second chromatography system, an UF / DF system, and a plurality of mixing vessels; the second configuration of the production lines is different from the first configuration of the production lines; a second plurality of control modules implemented for controlling operation of the second device; At least one mixing vessel of the plurality of mixing vessels is included in both the first configuration and the second configuration; 34. The automated system of claim 33, wherein the at least one mixing vessel has a first function in the first configuration and a second function in the second configuration, the second function being different from the first function. (35) a portable filter bank including a plurality of filter assemblies, a first filter assembly of the plurality of filter assemblies including a first filter and a second filter assembly of the plurality of filter assemblies including a second filter; A production facility control system, comprising: monitoring a pressure within the first filter assembly as material flows through the first filter assembly; determining that the pressure in the first filter assembly is at least a threshold value; and a production facility control system that sends a signal to operate a diverter valve coupled to the first filter assembly and the second filter assembly to cause material to flow into the second filter assembly. (36) 20. The automated equipment of claim 19, wherein the one or more disposable perfusion bioreactors are capable of containing a liquid medium under conditions that allow cultured cells to secrete the protein into the medium for a production culture period of at least 20 days. (37) The automated equipment according to (20), wherein the target protein is a recombinant protein. (38) The automated equipment according to (20), wherein the target protein is a therapeutic protein. (39) 1. A process for producing a purified protein drug substance comprising a protein of interest, comprising the steps of: (a) culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that permit the cells to secrete said protein into said liquid medium over a production culture period of at least 10 days, wherein during said production culture period, fresh sterile liquid medium is periodically or continuously added to the one or more disposable perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors in direct relation to a volume of culture that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed, and the volume of permeate removed is automatically and fluidly delivered from the one or more disposable perfusion bioreactors to a disposable surge vessel and then to a first chromatography system, whereby said protein is collected in a protein isolate fraction; (b) switching said protein isolate fraction to a low pH or detergent viral inactivation system, and optionally a neutralization system, to obtain a viral inactivation product pool comprising said protein; (c) introducing the viral inactivation product pool into a second chromatography system to obtain a purified product pool comprising the protein; (d) switching the purified product pool containing the protein to an optional third chromatography system and / or a viral filtration system to obtain a virus-free filtrate containing the protein; (e) switching the virus-free filtrate into an ultrafiltration / diafiltration system to obtain the purified protein drug substance comprising the protein of interest. (40) 39. The process of claim 39, wherein the fresh sterile liquid medium is mixed simultaneously from a plurality of different concentrated medium component solutions and aqueous diluents before being added to the one or more perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors. (41) 39. The process according to claim 39, wherein the protein of interest is a recombinant protein. (42) 39. The process according to claim 39, wherein the protein of interest is a therapeutic protein. (43) 1. An automated facility for producing a purified protein drug substance, said facility comprising: (a) one or more disposable perfusion bioreactors capable of containing a liquid culture medium under conditions that permit cultured mammalian cells to secrete a protein of interest into the medium over a production culture period of at least 10 days, the disposable perfusion bioreactors being adapted to fluidly receive fresh sterile liquid culture medium into each of the perfusion bioreactors in direct relation to a volume of conditioned medium that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed during the production culture period; (b) a first disposable surge vessel (SUSV1) into which the removed permeate volume is automatically and fluidly delivered from the one or more disposable perfusion bioreactors; (c) a first chromatography system adapted to automatically ...

Claims

1. A process for producing a purified protein of interest, comprising the steps of:

1. A process comprising: (a) culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that permit the cells to secrete said protein into said liquid medium over a production culture period of at least 10 days, wherein during said production culture period, fresh sterile liquid medium is periodically or continuously added to the one or more disposable perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors that is directly related to a volume of culture that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed, and wherein the volume of permeate removed is automatically and fluidly fed from the one or more disposable perfusion bioreactors to a disposable surge vessel and then to a first chromatography system, whereby said protein is collected in a protein isolate fraction.

2. A process for producing a purified protein of interest, comprising the steps of:

1. A process comprising: (a) culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that permit the cells to secrete said protein into said liquid medium over a production culture period of at least 10 days, wherein during said production culture period, periodically or continuously, fresh sterile liquid medium that is simultaneously mixed from a plurality of different concentrated medium component solutions and an aqueous diluent is added to the one or more disposable perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors in direct relation to a volume of culture that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed, and wherein the volume of permeate removed is automatically and fluidly fed from the one or more disposable perfusion bioreactors to a disposable surge vessel and then to a first chromatography system, whereby said protein is collected in a protein isolate fraction.

3. The following steps:

3. The process of claim 1 or claim 2, further comprising: (b) switching the protein isolate fraction to a low pH or detergent viral inactivation system, and optionally a neutralization system, to obtain a viral inactivation product pool comprising the protein.

4. The following steps: (c) introducing the viral inactivation product pool into a second chromatography system to obtain a purified product pool comprising the protein; (d) switching the purified product pool containing the protein to an optional third chromatography system and / or viral filtration system to obtain a virus-free filtrate containing the protein; 4. The process of claim 3, further comprising: (e) switching the virus-free filtrate to an ultrafiltration / diafiltration system to obtain a composition comprising a purified protein of interest.

5. 3. The process of claim 1 or claim 2, wherein the protein of interest is a recombinant protein.

6. 3. The process of claim 1 or claim 2, wherein the protein of interest is a therapeutic protein.

7. 5. The process of claim 1, claim 2, or claim 4, wherein one or more of the first chromatography system, the second chromatography system, the third chromatography system, the low pH or detergent viral inactivation system, the neutralization system, the viral filtration system, or the UF / DF system include one or more disposable components.

8. 3. The process of claim 1 or claim 2, wherein the mammalian cells are cultured in 2, 3, 4, 5 or 6 disposable perfusion bioreactors.

9. 3. The process of claim 1 or 2, wherein the one or more disposable bioreactors can contain a volume of about 50 L to about 4000 L of liquid medium.

10. 3. The process of claim 2, wherein the fresh sterile liquid medium is added to the one or more perfusion bioreactors by directly injecting the plurality of different concentrated medium component solutions in a fixed ratio relative to one another into the perfusion bioreactor, while an aqueous diluent is also added in varying ratios to the plurality of different concentrated component solutions to maintain a constant culture volume in each perfusion bioreactor.

11. 3. The process of claim 2, wherein the fresh sterile liquid medium is added to the one or more perfusion bioreactors by injecting the plurality of different concentrated medium component solutions and the aqueous diluent directly into the perfusion bioreactor in a fixed ratio relative to one another to maintain a constant culture volume in each perfusion bioreactor.

12. 3. The process of claim 2, wherein the fresh sterile liquid medium is added to the one or more perfusion bioreactors by injecting the plurality of different concentrated medium component solutions and the aqueous diluent in a fixed ratio relative to one another into a mixing chamber to maintain a constant culture volume, where fresh sterile liquid medium is mixed simultaneously before being added to each perfusion bioreactor.

13. 3. The process of claim 1 or 2, wherein an automatic control device including a detector is used to measure the fluid volume in the disposable surge receptacle, and a processor varies a pump speed of the first chromatography system to maintain a preset volume range in the disposable surge receptacle.

14. 5. The process of claim 3 or claim 4, wherein one or more of steps (b), (c), (d) or (e) are performed automatically and fluidly with uninterrupted flow from a previous step, a surge receptacle is used during one or more steps, and the processor varies the pump speed in a subsequent step to adjust a preset volume range of the surge receptacle preceding the subsequent step.

15. 15. The process of claim 14, wherein in-line or in-vessel adjustment of pH and / or conductivity loading is performed during one or more of steps (b), (c), (d), or (e).

16. (i) a process automation system in electronic communication with at least the one or more disposable perfusion bioreactors, the disposable surge vessel, and the first chromatography system; (ii) the process automation system stores a first set of control modules for controlling operation of at least one disposable perfusion bioreactor of the one or more disposable perfusion bioreactors; (iii) the process automation system stores a second set of control modules for controlling operation of the supply tank; (iv) the process automation system stores a third set of control modules for controlling operation of the collection tank; 3. The process of claim 1 or claim 2, wherein (v) the at least one disposable perfusion bioreactor is logically configured to be coupled to one or more feed tanks, one or more harvest tanks, or a filter bank.

17. 17. The process of claim 16, wherein the at least one disposable perfusion bioreactor is disposed on a skid, the skid comprising a plurality of communication interfaces for electronically coupling the at least one disposable perfusion bioreactor to a plurality of portable instruments.

18. determining, by the process automation system, that the disposable surge receptacle is coupled to one of the plurality of communication interfaces based on data received via the communication interface, the data indicating an identifier of the disposable surge receptacle and a function of the disposable surge receptacle; 20. The process of claim 17, further comprising: determining, based at least in part on the identifier and the function of the disposable surge receptacle, that the disposable surge receptacle is a collection tank and that the third set of control modules is for controlling operation of the disposable surge receptacle.

19. determining, by the process automation system, that a mixing vessel is coupled to an additional communication interface of the plurality of communication interfaces based on additional data received via an additional communication interface, the additional data indicating an additional identifier of the mixing vessel and an additional function of the mixing vessel; 20. The process of claim 18, further comprising: determining, based at least in part on the additional identifier and the additional function of the mixing vessel, that the mixing vessel is a supply tank and that the second set of control modules is for controlling operation of the mixing vessel.

20. 20. The process of claim 18, wherein the identifier of the disposable surge receptacle and the capabilities of the disposable surge receptacle are stored in a dongle coupled to the disposable surge receptacle.

21. 3. The process of claim 1 or claim 2, wherein the production culture period is at least 20 days.

22. 1. An automated facility for producing a purified protein of interest, said facility comprising: (a) one or more disposable perfusion bioreactors capable of containing a liquid medium under conditions that permit cultured cells to secrete said protein into the liquid medium during a production culture period of at least 10 days, the disposable perfusion bioreactors being adapted to fluidly receive fresh sterile liquid medium into each of said perfusion bioreactors in direct relation to a volume of conditioned medium that is continuously or periodically removed from each of said perfusion bioreactors as a volume of permeate or cell bleed during said production culture period; (b) a first disposable surge vessel (SUSV1) into which the removed permeate volume is automatically and fluidly delivered from the one or more disposable perfusion bioreactors; (c) a first chromatography system adapted to automatically and fluidly receive a cell-free permeate from said SUSV1, whereby said protein is captured in a protein isolate fraction; The automated equipment is controlled by a process automation system (PAS).

23. 23. The automated equipment of claim 22, further comprising a plurality of reservoirs each adapted to contain a concentrated media component solution or an aqueous diluent, the plurality of reservoirs being fluidly connected to the perfusion bioreactor either directly or indirectly via an optional mixing vessel adapted to receive the concentrated media component solution and the aqueous diluent in a predetermined ratio from the plurality of reservoirs and simultaneously mix them, the optional mixing vessel being fluidly connected directly to the perfusion bioreactor.

24. (d) a low pH or detergent viral inactivation system, and optionally a neutralization system, adapted to automatically and fluidly receive the protein isolate fraction from the first chromatography system, thereby obtaining a viral inactivation product pool comprising the protein; 23. The automated equipment of claim 22, further comprising: (e) a holding vessel or a second disposable surge vessel adapted to receive the viral inactivation product pool.

25. (f) a second chromatography system adapted to fluidly receive the viral inactivation product pool from the holding vessel or the second disposable surge vessel, thereby obtaining a purified product pool comprising the protein; and (g) an optional third chromatography system and / or virus filtration system adapted to fluidly receive the purified product pool comprising the protein from the second chromatography system, thereby obtaining a virus-free filtrate comprising the protein; and 23. The automated equipment of claim 22, further comprising: (h) an UF / DF system adapted to fluidly receive the virus-free filtrate from the second chromatography system, or from the third chromatography system and / or the virus filtration system, thereby obtaining the purified protein of interest.

26. 23. The automated equipment of claim 22, wherein the one or more disposable perfusion bioreactors are capable of containing a volume of from about 50 L to about 4000 L of liquid medium.

27. 23. The automated equipment of claim 22, further comprising an automatic control device including a detector for measuring a fluid volume in SUSV1 and a processor for varying a pump speed of the first chromatography system to maintain a preset volume range in SUSV1.

28. 23. The automated equipment of claim 22, wherein one or more of the first chromatography system, the second chromatography system, the third chromatography system, the low pH or detergent viral inactivation system, the neutralization system, the viral filtration system, or the UF / DF system comprise disposable components.

29. directly fluidly connected downstream of the first chromatographic system; (i) a second disposable surge receptacle; or (ii) at least two automatically switchable alternative disposable collection vessels (SUCV1 and SUCV2) adapted to receive said protein isolate fractions; 23. The automated equipment of claim 22, wherein (i) and (ii) are adapted to receive the protein isolate fraction from the first chromatography system and fluidly deliver the protein isolate fraction to the low pH or detergent viral inactivation system.

30. The low pH or detergent viral inactivation system, and optionally the neutralization system, (i) a second disposable surge vessel adapted to receive the protein isolate fraction; or (ii) at least two automatically switchable alternative disposable collection vessels (SUCV1 and SUCV2) adapted to receive said protein isolate fractions; 25. The automated installation of claim 24, wherein viral inactivation, and optionally neutralization, takes place in the second disposable surge vessel, or in SUCV1 and SUCV2.

31. 23. The automated equipment of claim 22, further comprising a hollow fiber membrane, a series of depth filters, or a filtration cart before the permeate is automatically and fluidly delivered to the SUSV1.

32. 25. The automated equipment of claim 24, further comprising in (e) a disposable surge receptacle adapted to receive the viral inactivation product pool.

33. The automated equipment of claim 22, further comprising a heat exchanger upstream of the SUSV1.

34. 23. The automated equipment of claim 22, further comprising a filtration system upstream of the SUSV1.

35. (i) the second chromatography system; (ii) any third chromatographic system; (iii) the virus filtration system, and (iv) the UF / DF system, wherein one or more of:

26. The automated installation of claim 25, automatically and fluidly connected to a preceding system, and optionally using a surge receptacle to regulate the uninterrupted flow of material between said connected systems.

36. the at least one or more disposable perfusion bioreactors, SUSV1, the first chromatography system, the low pH or detergent viral inactivation system, the holding vessel or disposable surge vessel, the second chromatography system, the optional third chromatography system and / or viral filtration system, and the UF / DF system comprise a first device arranged in a first configuration of a production line for the purified protein of interest; 26. The automated installation of any of claims 22 to 25, wherein a first plurality of control modules are implemented to control the operation of the first equipment.

37. A second piece of equipment is disposed in a second configuration of a production line for additional purified proteins of interest, the second configuration of the production line including at least one or more disposable perfusion bioreactors, a first chromatography system, a low pH or detergent viral inactivation system, a second chromatography system, an UF / DF system, and a plurality of mixing vessels; the second configuration of the production lines is different from the first configuration of the production lines; a second plurality of control modules implemented for controlling operation of the second device; at least one mixing vessel of the plurality of mixing vessels is included in both the first configuration and the second configuration; 37. The automated system of claim 36, wherein the at least one mixing vessel has a first function in the first configuration and a second function in the second configuration, the second function being different from the first function.

38. a portable filter bank including a plurality of filter assemblies, a first filter assembly of the plurality of filter assemblies including a first filter and a second filter assembly of the plurality of filter assemblies including a second filter; A production facility control system, comprising: monitoring a pressure within the first filter assembly as material flows through the first filter assembly; determining that the pressure in the first filter assembly is at least a threshold value; 26. The automated facility of claim 22, further comprising: a production facility control system that sends a signal to operate a diverter valve coupled to the first filter assembly and the second filter assembly to cause material to flow into the second filter assembly.

39. 40. The automated equipment of claim 38, wherein during a first time period, the filter bank is coupled between the first disposable surge vessel (SUSV1) and the first chromatography system, the material comprises the permeate, and the filter bank is coupled to a first dongle indicating a first identifier of the filter bank and a first function of the filter bank.

40. 40. The automated equipment of claim 39, wherein during a second period of time, the filter bank is included in the UF / DF system, the material is the virus-free filtrate, and the filter bank is coupled to a second dongle indicating a second identifier of the filter bank and a second function of the filter bank.

41. 23. The automated equipment of claim 22, wherein the one or more disposable perfusion bioreactors are capable of containing liquid culture medium under conditions that allow cultured cells to secrete the protein into the medium for a production culture period of at least 20 days.

42. 23. The automated equipment of claim 22, wherein the protein of interest is a recombinant protein.

43. 23. The automated equipment of claim 22, wherein the protein of interest is a therapeutic protein.

44. 1. A process for producing a purified protein drug substance comprising a protein of interest, comprising the steps of: (a) culturing mammalian cells in one or more disposable perfusion bioreactors containing a liquid medium under conditions that permit the cells to secrete said protein into said liquid medium over a production culture period of at least 10 days, wherein during said production culture period, fresh sterile liquid medium is periodically or continuously added to the one or more disposable perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors in direct relation to a volume of culture that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed, and the volume of permeate removed is automatically and fluidly fed from the one or more disposable perfusion bioreactors to a disposable surge vessel and then to a first chromatography system, whereby said protein is collected in a protein isolate fraction; (b) switching said protein isolate fraction to a low pH or detergent viral inactivation system, and optionally a neutralization system, to obtain a viral inactivation product pool comprising said protein; (c) introducing the viral inactivation product pool into a second chromatography system to obtain a purified product pool comprising the protein; (d) switching the purified product pool containing the protein to an optional third chromatography system and / or viral filtration system to obtain a virus-free filtrate containing the protein; (e) switching the virus-free filtrate into an ultrafiltration / diafiltration system to obtain the purified protein drug substance comprising the protein of interest.

45. 45. The process of claim 44, wherein the fresh sterile liquid medium is mixed simultaneously from a plurality of different concentrated medium component solutions and aqueous diluents before being added to the one or more perfusion bioreactors to maintain a constant culture volume in each of the perfusion bioreactors.

46. 45. The process of claim 44, wherein the protein of interest is a recombinant protein.

47. 45. The process of claim 44, wherein the protein of interest is a therapeutic protein.

48. 1. An automated facility for producing a purified protein drug substance, said facility comprising: (a) one or more disposable perfusion bioreactors capable of containing a liquid culture medium under conditions that permit cultured mammalian cells to secrete a protein of interest into the medium over a production culture period of at least 10 days, the disposable perfusion bioreactors being adapted to fluidly receive fresh sterile liquid culture medium into each of the perfusion bioreactors in direct relation to a volume of conditioned medium that is continuously or periodically removed from each of the perfusion bioreactors as a volume of permeate or cell bleed during the production culture period; (b) a first disposable surge vessel (SUSV1) into which the removed permeate volume is automatically and fluidly delivered from the one or more disposable perfusion bioreactors; (c) a first chromatography system adapted to automatically and fluidly receive permeate from said SUSV1, whereby said protein is captured in a protein isolate fraction; (d) a low pH or detergent viral inactivation system, and optionally a neutralization system, adapted to automatically and fluidly receive the protein isolate fraction from the first chromatography system, thereby obtaining a viral inactivation product pool comprising the protein; (e) a holding vessel or disposable surge vessel adapted to receive the viral inactivation product pool; (f) a second chromatography system adapted to fluidly receive the viral inactivation product pool from the holding vessel or the disposable surge vessel, thereby obtaining a purified product pool comprising the protein; and (g) an optional third chromatography system and / or virus filtration system adapted to fluidly receive the purified product pool comprising the protein from the second chromatography system, thereby obtaining a virus-free filtrate comprising the protein; and (h) an UF / DF system adapted to fluidly receive the virus-free filtrate from the second chromatography system, or from the third chromatography system and / or the virus filtration system, thereby obtaining the purified protein drug substance; The automated equipment is controlled by a process automation system (PAS).

49. 49. The automated equipment of claim 48, wherein a plurality of reservoirs, each adapted to contain a concentrated media component solution or an aqueous diluent, are fluidly connected to the perfusion bioreactor either directly or indirectly via an optional mixing vessel adapted to receive the concentrated media component solutions and the aqueous diluent in predetermined ratios from the plurality of reservoirs and mix them simultaneously, the optional mixing vessel being fluidly connected directly to the perfusion bioreactor.

50. 49. The automated equipment of claim 48, wherein the protein of interest is a recombinant protein.

51. 49. The automated equipment of claim 48, wherein the protein of interest is a therapeutic protein.

52. 49. The automated installation of any of claims 22 to 25 or claim 48, wherein the installation is configured to operate in a continuous manner.

53. 45. The process of any of claims 1 to 4, claim 7, or claim 44, wherein the process is carried out in a continuous mode.

54. 45. The process of any of claims 1 to 4, claim 7, or claim 44, wherein the first chromatographic system is disinfected with a chemical disinfectant solution comprising peracetic acid prior to use.

55. 45. The process of claim 4 or claim 44, wherein the UF / DF system comprises a single pass tangential flow filtration (SPTFF) and the operating pressure of the SPTFF is controlled in the range of about 0.25 psi to about 60 psi.

56. 45. The process of claim 4 or claim 44, wherein the UF / DF system comprises an in-line depth filtration (ILDF), and the operating pressure of the ILDF is controlled in the range of about 0.25 psi to about 60 psi.

Images