Continuous production of recombinant proteins

Abstract

To provide methods and systems for continuous production of recombinant proteins.SOLUTION: A method comprises: (a) capturing a recombinant protein from a substantially cell-free sample using one or more capture chromatography systems and eluting the recombinant protein from the one or more capture chromatography systems to produce eluates comprising the recombinant protein, where the eluates are homogenized into a single mixture comprising the recombinant protein; (b) subjecting the homogenized single mixture to one or more post-capture chromatography systems and collecting product output comprising the recombinant protein; and (c) subjecting the product output to ultrafiltration and diafiltration to purify the recombinant protein. The method is implemented integrally and continuously from the step (a) to the step (c).SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 828,755, filed April 3, 2019, the disclosure of which is expressly incorporated herein by reference.

[0002] The present disclosure relates to methods and systems for the continuous production of recombinant proteins. In certain embodiments, the present disclosure relates to methods and systems for the continuous production of recombinant proteins using capture chromatography, post-capture chromatography, viral filtration, and ultrafiltration / diafiltration. [Background technology]

[0003] Continuous manufacturing is a common production method in industries such as petrochemical and food production. The advantages of continuous processes include steady operation, compact equipment, high volumetric productivity, streamlined process flow, low cycle times, and reduced capital costs. Non-Patent Document 1. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Konstantinov et al., J. Pharm. Sci. 104(3):813-20 (2015) Summary of the Invention [Problem to be solved by the invention]

[0005] Continuous manufacturing has not been widely implemented in the biopharmaceutical industry. The technical needs of upstream and downstream development are very different, and there is limited experience with continuous downstream operation. Therefore, the biopharmaceutical industry needs integrated continuous bioprocesses that can run for extended periods with minimal operator interaction. [Means for solving the problem]

[0006] Provided herein is a method for continuously producing a recombinant protein, the method comprising: (a) capturing a recombinant protein from a substantially cell-free sample using one or more capture chromatography systems and eluting the recombinant protein from the one or more capture chromatography systems to generate an eluate comprising the recombinant protein, wherein the single or multiple eluates are homogenized into a single mixture comprising the recombinant protein; (b) subjecting the single mixture to one or more post-capture chromatography systems and collecting a product output comprising the recombinant protein; and (c) subjecting the product output to ultrafiltration and diafiltration to purify the recombinant protein, the method being performed in an integrated and continuous manner from step (a) to step (c).

[0007] Also provided herein is a manufacturing system for producing a recombinant protein, the manufacturing system including: (a) a first unit operation including a bioreactor including host cells that produce the recombinant protein; (b) a second unit operation including one or more capture chromatography systems; (c) a third unit operation including one or more post-capture chromatography systems; and (d) a fourth unit operation including an ultrafiltration system and a diafiltration system.

[0008] Also provided herein is a method for continuously producing a recombinant protein, comprising: (a) capturing a recombinant protein from a substantially cell-free sample using one or more capture chromatography systems and eluting the recombinant protein from the one or more capture chromatography systems to generate an eluate comprising the recombinant protein, which eluate is homogenized into a single mixture comprising the recombinant protein; (b) subjecting the single homogenized mixture to viral inactivation; (c) subjecting the single homogenized mixture of step (b) to one or more post-capture chromatography systems and collecting a product output comprising the recombinant protein; (d) subjecting the product output of step (c) to viral filtration; and (e) subjecting the product output of step (d) to ultrafiltration and diafiltration to purify the recombinant protein, wherein the method is performed in an integrated and continuous manner from step (a) to step (e).

[0009] Other aspects, embodiments, and implementations will become apparent from the following detailed description and claims, taken together with the accompanying drawings as appropriate. [Brief description of the drawings]

[0010] [Figure 1] 1 is a schematic diagram showing the process flow of the method disclosed herein, which includes: (1) recombinant protein production in a bioreactor coupled with a dual alternating tangential flow (ATF) filter and cell separation (denoted as "BRX"), (2) capturing recombinant protein using one or more capture chromatography systems and capturing recombinant protein present in the culture medium obtained from the bioreactor connected to the capture chromatography system (denoted as "Capture"), (3) viral inactivation of the medium (denoted as "VI"), (4) subjecting the medium to one or more post-capture chromatography systems (denoted as "Post-Capture"), (5) viral filtration of the medium, and (6) UF / DAF of the medium, a continuous process for recombinant protein production with in-line buffer. [Diagram 2] 2A-2B are diagrams outlining the integration of unit operations of the methods and systems disclosed herein. FIG. 2A shows the duration of the integration of unit operations for continuous operation of each unit operation. FIG. 2B shows the average protein residence time of each unit operation. The methods and systems disclosed herein produced approximately 5 kg of drug substance in 25 days. [Figure 3-1] Figures 3A-3F are graphs showing the operational performance of Protein A of the methods and systems disclosed herein: Figure 3A shows the concentration (g / L) of Protein A eluate, Figure 3B shows the Protein A eluate residual host cell protein, Figure 3C shows the Protein A eluate residual protein, Figure 3D shows the Protein A inlet mass flow rate, and Figures 3E and 3F show the Protein A UV chromatograms. [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Figure 4-1] Figures 4A-4G are graphs showing the viral inactivation operational performance of the methods and systems disclosed herein: Figure 4A shows the viral inactivation pH maintained throughout the unit operation, Figure 4B shows the protein concentration before inactivation, Figure 4C shows the protein mass flow rate during viral inactivation, Figure 4D shows the acid flow rate during viral inactivation, Figure 4E shows the base flow rate during viral inactivation, Figure 4F shows the high molecular weight species during viral inactivation, and Figure 4G shows the protein concentration after viral inactivation. [Figure 4-2] Continued from Figure 4-1. [Figure 4-3] Continued from Figure 4-2. [Figure 5-1] Figures 5A-5D are graphs showing post-capture operational performance of the methods and systems disclosed herein: Figure 5A shows residual protein A in the post-capture eluate, Figure 5B shows protein concentration in the post-capture eluate, Figure 5C shows a UV chromatogram, and Figure 5D shows residual host cell protein in the post-capture eluate. [Figure 5-2] Continued from Figure 5-1. [Figure 6-1]Figure 6A is a schematic diagram of virus filtration of the methods and systems disclosed herein. Figures 6B-6G are graphs showing the virus filtration operational performance of the methods and systems disclosed herein. Figure 6B shows protein concentration after virus filtration, Figure 6C shows high molecular weight species after virus filtration, Figure 6D shows protein mass flux during virus filtration, Figure 6E shows filtrate volume flux during virus filtration, Figure 6F shows transmembrane pressure during virus filtration, and Figure 6G shows filter performance during virus filtration. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. [Figure 6-4] Continued from Figure 6-3. [Figure 7-1] Figures 7A-7E are graphs showing ultrafiltration operational performance of the methods and systems disclosed herein: Figure 7A shows protein concentration and conversion after ultrafiltration, Figure 7B shows filter performance during ultrafiltration, Figure 7C shows inlet protein mass flux during ultrafiltration, Figure 7D shows permeate mass flux during ultrafiltration, and Figure 7E shows transmembrane pressure during ultrafiltration. [Figure 7-2] Continued from Figure 7-1. [Figure 8-1] Figures 8A-8E are graphs illustrating the diafiltration operational performance of the methods and systems disclosed herein: Figure 8A shows the permeate volume flux during diafiltration, Figure 8B shows the protein mass flux during diafiltration, Figure 8C shows the transmembrane pressure during diafiltration, Figure 8D shows the diafiltration ratio, and Figure 8E shows the filter performance during diafiltration. [Figure 8-2] Continued from Figure 8-1. [Figure 8-3] Continued from Figure 8-2. [Figure 9-1]Figures 9A-H are graphs showing properties of drug substances produced using the methods and systems disclosed herein: Figure 9A shows the drug substance concentration, Figure 9B shows the high molecular weight species of the drug substance, Figure 9C shows the charge profile of the drug substance, Figure 9D shows the osmolality of the drug substance, Figure 9E shows the pH of the drug substance, Figure 9F shows the host cell protein measured by ELISA of the drug substance, Figure 9G shows the non-reduced purity of the drug substance, and Figure 9H shows the residual protein A of the drug substance. [Figure 9-2] Continued from Figure 9-1. [Figure 9-3] Continued from Figure 9-2. [Figure 9-4] Continued from Figure 9-3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure relates to a method and system for continuous production of recombinant proteins. In certain embodiments, the present disclosure relates to a method and system that uses capture chromatography, post-capture chromatography, and ultrafiltration / diafiltration to produce recombinant proteins. The methods and systems described herein provide for continuous and time-efficient production of recombinant proteins.

[0012] As utilized in accordance with this disclosure, unless otherwise indicated, all technical and scientific terms shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0013] In some embodiments, a method for continuously producing a recombinant protein includes capturing a recombinant protein from a substantially cell-free sample using one or more capture chromatography systems, eluting the recombinant protein from the one or more capture chromatography systems to generate an eluate comprising the recombinant protein, wherein the eluate is homogenized into a single mixture comprising the recombinant protein, and subjecting the homogenized single mixture to one or more post-capture chromatography systems to generate an eluate comprising the recombinant protein. Provided herein is an integrated, continuous process that includes collecting the product output and subjecting the product output to ultrafiltration and diafiltration to purify the recombinant protein.

[0014] Also provided herein is a manufacturing system for producing a recombinant protein, the manufacturing system comprising a first unit operation comprising a bioreactor comprising host cells that produce the recombinant protein, a second unit operation comprising one or more capture chromatography systems, a third unit operation comprising one or more post-capture chromatography systems, and a fourth unit operation comprising an ultrafiltration system and a diafiltration system.

[0015] In some embodiments, the manufacturing system includes a fifth unit operation located between the first unit operation and the second unit operation, the fifth unit operation including a subsystem for preliminary viral inactivation. In some embodiments, the manufacturing system includes a sixth unit operation located between the third unit operation and the fourth unit operation, the sixth unit operation including a second subsystem for preliminary viral filtration. In some embodiments, the manufacturing system includes a seventh unit operation including a third subsystem, the third subsystem including an in-line excipient for formulating a therapeutic drug substance.

[0016] As used herein, "substantially cell-free" refers to a sample that is at least or about 90% free (e.g., at least or about 95%, 96%, 97%, 98%, or at least or about 99%, or about 100% free) of a particular substance, such as mammalian cells.

[0017] In certain embodiments, a substantially cell-free sample is removed from a perfusion bioreactor comprising host cells producing a recombinant protein, a fed-batch bioreactor comprising host cells producing a recombinant protein, or a clarified liquid culture comprising host cells producing a recombinant protein.

[0018] As used herein, "liquid culture medium" refers to a liquid that contains sufficient nutrients for cells to grow or proliferate in vitro. For example, the liquid culture medium can include one or more of: amino acids (e.g., the 20 amino acids), purines (e.g., hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvic acid, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate. In some embodiments, the liquid culture medium can include serum from a mammal. In some embodiments, the liquid culture medium does not include serum or other extracts from a mammal (defined liquid culture medium). In some embodiments, the liquid culture medium can include trace metals, mammalian growth hormones, and / or mammalian growth factors. Additional suitable liquid culture media are known in the art and are commercially available.

[0019] As used herein, a "perfusion bioreactor" refers to a bioreactor that contains a plurality of cells in a first liquid culture medium, and culturing the cells present in the bioreactor includes periodically or continuously removing the first liquid culture medium and simultaneously or immediately thereafter adding a substantially equal amount of a second liquid culture medium to the bioreactor. In some embodiments, there is a gradual change (e.g., increase or decrease) in the amount of the first liquid culture medium that is removed and added over a gradual period of time during the culture period (e.g., a period of about 24 hours, a period between about 1 minute and about 24 hours, or a period of more than 24 hours) (e.g., a medium replenishment rate on a daily basis, etc.). The percentage of medium removed and replaced each day can vary depending on the particular cells being cultured, the initial seeding density, and the cell density at a particular time.

[0020] As used herein, "fed-batch bioreactor" refers to a bioreactor containing a plurality of cells in which nutrients required for cell growth and product formation are supplied intermittently or continuously via one or more feed streams.

[0021] As used herein, "clarified liquid culture medium" refers to a liquid culture medium obtained from a bacterial or yeast cell culture and that is substantially free of bacterial or yeast cells (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% free).

[0022] As used herein, "recombinant protein" refers to an immunoglobulin, a protein fragment, an engineered protein, a blood factor, a nanobody, or an enzyme, and therefore also includes an antibody or an antibody fragment.

[0023] As used herein, a "unit operation" refers to a functional step that can be performed in a method for producing a recombinant protein, or in a component of a system used in a recombinant protein production process. For example, a unit operation can be filtration (e.g., removal of contaminating bacteria, yeast viruses, or mycobacteria, and / or specific substances from a liquid containing the recombinant therapeutic protein), capture, removal of an epitope tag, purification, retention or preservation, polishing, viral inactivation, adjustment of the ionic concentration and / or pH of a liquid containing the recombinant protein, and removal of unwanted salts.

[0024] As used herein, the term "continuous process" or "continuous system" refers to a process or system in which at least some of the unit operations are continuously supplied with liquid. A unit operation is continuous if it can process a continuous flow input over time. A continuous unit operation has minimal internal retention. The output is continuous or discretized in small packets that are produced periodically. A process is continuous if it consists of integrated (physically connected) consecutive unit operations with zero or minimal internal retention between them.

[0025] As used herein, an "integrated method" refers to a method carried out using structural elements that function in concert to achieve a particular result (e.g., production of a recombinant protein from a liquid culture medium).

[0026] As used herein, "eluate" refers to the liquid exiting a chromatography column or membrane that contains detectable amounts of recombinant protein.

[0027] As used herein, "chromatographic media" refers to the material that is packed into a chromatography column.

[0028] In some embodiments, the systems disclosed herein are closed systems. Closed systems include unit operations that are designed and operated to limit exposure to the outside environment. Materials can be introduced into the closed system, but additions must be made to avoid exposing the product to the indoor environment.

[0029] The methods and systems disclosed herein include capturing a recombinant protein using one or more capture chromatography systems and eluting the recombinant protein from the one or more capture chromatography systems to generate an eluate that includes the recombinant protein. The chromatography media included in one or more of the capture chromatography systems may be a capture mechanism (e.g., a Protein A-bound capture mechanism, a Protein G-bound capture mechanism, an antibody or antibody fragment-bound capture mechanism, a substrate-bound capture mechanism, a cofactor-bound capture mechanism, or a combination thereof). The capture chromatography system or systems may be resins that utilize a capture mechanism (e.g., a tag-binding capture mechanism, and / or an aptamer-binding capture mechanism). In some embodiments, the one or more capture chromatography systems include a chromatography medium made of resin beads, porous membranes, or nanofibers. In some embodiments, the chromatography medium is functionalized for affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, or mixed-mode chromatography. In some embodiments, the affinity chromatography medium is a Protein A-based resin.

[0030] In some embodiments, the one or more capture chromatography systems are cyclic countercurrent chromatography systems (PCCS). In some embodiments, the PCCS includes two chromatography columns or includes a modified AKTA system (GE Healthcare, Piscataway, NJ) that can run up to, for example, 4, 5, 6, 7, 8, or more columns. In some embodiments, the PCCS utilizes a column switch mechanism. A column switch event is triggered by the detection of a level of recombinant protein detected by UV absorbance corresponding to a certain level of recombinant protein in the liquid passing through the chromatography system, a specific volume of liquid (e.g., buffer), or a specific time lapse.

[0031] To capture a recombinant protein using a capture chromatography system, a series of chromatographic steps must be performed: loading, washing, elution and regeneration of the capture chromatography system.

[0032] In some embodiments, the eluate from the one or more capture chromatography systems is homogenized into a single mixture containing the recombinant protein. In some embodiments, the homogenized single mixture is subjected to one or more post-capture chromatography systems, and a product output containing the recombinant protein is collected. The post-capture chromatography system is used to remove remaining trace or minor contaminants or impurities from the liquid containing the recombinant protein close to the final desired purity. In some embodiments, the one or more post-capture chromatography systems include a chromatography medium made of resin beads, porous membranes, or nanofibers. In some embodiments, the chromatography medium is functionalized for affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, or mixed-mode chromatography. In some embodiments, the one or more post-capture chromatography columns are cyclic countercurrent chromatography systems (PCCS).

[0033] Capturing a recombinant protein using a post-capture chromatography system requires a series of chromatographic steps: loading, washing, elution and regeneration of the post-capture chromatography system.

[0034] In some embodiments, the method and system includes ultrafiltration (UF) and / or diafiltration (DF) to further purify and concentrate the recombinant protein. UF / DF can not only increase the concentration of the recombinant protein but also replace buffer salts with buffers for certain formulations. Ultrafiltration (UF) is a type of membrane filtration in which hydrostatic pressure forces a liquid against a semipermeable membrane. In some embodiments, UF is performed with tangential flow filtration (TFF), including single TFF and high performance tangential flow filtration (HPTFF). Single pass tangential flow filtration (SPTFF) refers to any TFF system in which the conversion (permeate flow rate divided by inlet feed flow rate) in a single pass through the module is large enough that the system can operate without a retentate recycle loop. SPTFF is a method for, for example, chromatographic purification of a chromatographically purified ... It can be used in-line between other unit operations to reduce process volumes prior to purification or precipitation steps. Zydney, Biotechnol. Bioeng. 113(3):465-75, (2016).

[0035] As used herein, "diafiltration" refers to a method of using ultrafiltration membranes to remove, replace, or reduce the concentration of salts and buffer components from solutions containing proteins, such as antibodies, peptides, nucleic acids, and other biomolecules. Continuous diafiltration (also called constant volume diafiltration) involves washing out salts (or other low molecular weight species) of the original buffer in the retentate by adding new buffer, such as water or formulation buffer, to the retentate to form a formulation containing recombinantly produced polypeptides. Typically, new buffer is added at the same rate as filtrate is produced so that the volume of the retentate and the concentration of the product do not change significantly during diafiltration. In certain embodiments, diafiltration is performed using a single pass diafiltration cassette.

[0036] In some embodiments, the homogenized single mixture is subjected to viral inactivation prior to being subjected to one or more post-capture chromatographic systems. In some embodiments, viral inactivation includes solvent-detergent solution inactivation, heat inactivation, or acidic pH inactivation.

[0037] Solvent-detergent viral inactivation involves subjecting a sample containing the recombinant protein to an organic solvent and a detergent, which can be any solvent-detergent combination known to those of skill in the art, such as tri-n-butyl phosphate and Triton X-100™, Tween 80™ and sodium cholate, and others.

[0038] Viral inactivation by heat inactivation involves exposing a sample containing the recombinant protein to high temperatures. In some embodiments, the method involves heating the sample to temperatures of 45°C or higher, 46°C or higher, 47°C or higher, 48°C or higher, and up to about 49°C or higher, 50°C or higher, 51°C or higher, and higher. In some embodiments, the sample is heated to a temperature between 45°C and 65°C.

[0039] The period for which the sample is heated can vary. For example, in some embodiments, the sample is heated to the target temperature for a period between 1 minute and 6 hours. In some embodiments, the sample is heated to the target temperature for a period between 10 minutes and 180 minutes, between 20 minutes and 180 minutes, between 20 minutes and 60 minutes, or between 20 minutes and 40 minutes.

[0040] In some embodiments, inactivation by acidic pH is performed by adjusting the homogenized single mixture to a low pH in-line with one or more solutions and incubating the adjusted homogenized single mixture at the low pH while it becomes a viral inactivation mixture.

[0041] The methods and systems disclosed herein include in-line monitoring, including detection of recombinant protein levels detected by UV absorbance, detection of flow rate, and / or detection of liquid (e.g., buffer) volume. Monitoring of recombinant protein concentration (e.g., monitoring performed by UV monitoring) can be determined by any tool capable of in-line measurement of product concentration with feedback control.

[0042] In some embodiments, the homogenized single mixture is exposed to low pH for between 15 minutes and 2 hours. In certain embodiments, the low pH is between pH 3 and 5. The selection of the pH level depends on the stability profile of the recombinant protein and other buffer components. After DNA inactivation, the pH of the antibody solution can be adjusted to a more neutral pH, for example between 4.0 and 8.5, before continuing with the method.

[0043] In some embodiments, viral inactivation is performed in a tubular flow reactor such as that described in Parker et al., Biotechnol. Bioeng. 115(3):606-16 (2018). In some embodiments, the tubular flow reactor has a defined minimum residence time of at least 30 minutes.

[0044] In some embodiments, a virus removal step such as virus filtration is included after collection of the product output from the post-capture chromatography system. The virus removal step is performed to remove small non-enveloped viruses that are more resistant to virus inactivation treatment. In some embodiments, the virus filtration is performed in a pressurized loop, which includes a pressure vessel, a virus removal filter, and a sterilization filter. In some embodiments, the virus removal filter in the pressure loop is a hollow fiber virus filter. In some embodiments, the pressure vessel is a single-use, closed, and sterilizable container. As used herein, the term "sterilizable" refers to a container formed from a material that is compatible with known sterilization methods. In some embodiments, the virus filtration is performed using dead-end filtration. As used herein, "dead-end filtration" refers to filtration in which the entire liquid stream to be filtered passes through the filter without a recycle or retentate stream. In some embodiments, the virus removal filter is an ultrafiltration membrane or a nanofilter. One example of a suitable virus filtration system is disclosed in International Publication WO2018 / 035116, which is incorporated herein by reference.

[0045] In some embodiments, the first unit operation includes an outlet connected to an inlet of a second unit operation, the second unit operation includes an outlet connected to an inlet of a third unit operation, and the third unit operation includes an outlet connected to an inlet of a fourth unit operation. In some embodiments, the first unit operation includes an outlet connected to an inlet of a fifth unit operation, the fifth unit operation includes an outlet connected to an inlet of a second unit operation, the second unit operation includes an outlet connected to an inlet of a third unit operation, the third unit operation includes an outlet connected to an inlet of a sixth unit operation, and the sixth unit operation includes an outlet connected to an inlet of the fourth unit operation.

[0046] In some embodiments, the systems disclosed herein include a surge receptacle between each of the first unit operation, the second unit operation, the third unit operation, and the fourth unit operation, which can hold any liquid culture before moving to the next unit operation.

[0047] The systems described herein can also include liquid conduits disposed between any unit operations. Suitable liquid conduits are polyethylene, polycarbonate, or plastic tubes. The liquid conduits can also include any combination of one or more of the following: one or more in-line buffer adjustment reservoirs in liquid communication with the liquid conduits and arranged such that buffer stored in the in-line buffer adjustment reservoirs is added to the liquid present in the liquid conduits; and one or more filters arranged within the liquid conduits to filter (e.g., remove bacteria) the liquid present in the liquid conduits.

[0048] In some embodiments, the systems provided herein include a pump system that can include one or more of the following: one or more pumps known in the art, one or more filters known in the art, and one or more UV detectors.

[0049] In some embodiments, one or more excipients are produced after UF / DF. In addition to producing output, therapeutic drug substances are produced.

[0050] As used herein, a "therapeutic drug substance" refers to a substance, including recombinant proteins, that has been sufficiently purified or separated from contaminating proteins, lipids, nucleic acids (e.g., contaminating proteins, lipids, nucleic acids present in a liquid culture medium), host cells (e.g., from mammalian, yeast, or bacterial host cells), and biological contaminants (e.g., viral and bacterial contaminants) that it can be formulated into a pharmaceutical product without further substantial purification and / or decontamination steps.

[0051] In some embodiments, the systems disclosed herein are on skids. As used herein, "skid" refers to a three-dimensional solid structure that can serve as a platform or support for the systems described herein. A skid can provide mobility to a system or part thereof if it includes one or more structures that allow it to move (e.g., wheels, rollers, etc.).

[0052] In some embodiments, the methods and systems have a recovery rate of recombinant protein of at least about 40%, 50%, 55%, or 60%, up to about 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the purification recovery rate is at least about 60% to about 70%.

[0053] In some embodiments, the methods and systems described herein provide a net yield of recombinant protein in a therapeutic protein drug substance of at least about 5 g / day, 10 g / day or 20 g / day, 30 g / day or 40 g / day, and up to at least about 200 g / day, 300 g / day, 400 g / day, 500 g / day, or 1000 g / day, for a continuous period of at least about 5 days, 10 days, 20 days, or 30 days, and up to at least about 280 days, 290 days, 300 days, 310 days, 320 days, 330 days, 340 days, 350 days, or 365 days.

[0054] The following examples illustrate specific embodiments of the present disclosure and various uses thereof, which are provided for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way. EXAMPLES

[0055] Continuous production of recombinant proteins Recombinant monoclonal antibodies (mAbs) were produced over a 70-day period using a 100 L intensive perfusion bioreactor coupled with dual ATFs. The cell-free mAb-containing medium (harvest) was continuously pumped into a 100 L harvest surge vessel for further processing.

[0056] Antibody-containing media was captured using two pre-packed and irradiated Protein A affinity chromatography columns, 1 L (8 × 20 cm), in bind and elute MCC mode on a pilot-scale steam-in-place PCC skid. The capture step was run such that one of the two columns was always loaded from a harvest surge vessel, matching the average inlet flow rate of the capture step with the average outlet flow rate of the perfusion bioreactor. Loading of the Protein A column was controlled by Delta UV to approximately 3% breakthrough. The eluates of the individual capture columns were collected in a 5 L mixing vessel prior to viral inactivation. Protein A operation was integrated with the perfusion bioreactor on day 12 and run continuously for 58 days. Data summarizing the Protein A operational performance are shown in Figures 3A-3F.

[0057] The individual Protein A eluates were combined in a 5 L mixing vessel to ensure uniform flow for the viral inactivation unit operation. The outflow from the mixing vessel was then removed prior to the next Protein A elution cycle. The system was automated so that the entire vessel was processed at once. The homogenized Protein A eluate was adjusted in-line to a target pH of 3.6 by adding 1 M acetic acid. The low pH adjusted Protein A eluate was then pumped out of the tubular flow reactor (TFR) with a defined minimum residence time of at least 30 minutes to allow sufficient time at low pH to fully inactivate the viruses. The virus-inactivated Protein A eluate was then adjusted in-line to a target pH of 4.5 by adding 0.75 M sodium acetate. The adjusted virus-inactivated Protein A eluate was then treated with a sterilizing grade filter and continuously collected in a small surge vessel for further processing. The virus inactivation run was integrated with the preceding run on day 15 and was run continuously for 55 days. Data summarizing the virus inactivation run performance are shown in Figures 4A-4G.

[0058] The conditioned virus-inactivated Protein A eluate was further processed in two orthogonal chromatography runs using mixed-mode anion exchange and hydrophobic interaction chromatography resins, operated in flow-through mode. In this example run, there was no inter-step coordination between the two post-capture chromatography runs, and the flow-through from the mixed-mode column could be loaded directly onto the hydrophobic interaction column. The post-capture apparatus consisted of two 0.2 L (5 × 10 cm) pre-packed mixed-mode columns and two 0.2 L (5 × 10 cm) pre-packed hydrophobic interaction columns, operated in MCC mode on the same PCC skid as the Protein A runs. The conditioned virus-inactivated Protein A eluate stream was continuously loaded into one of two parallel post-capture apparatus, and column loading was automated such that approximately two masses of Protein A eluate were loaded in each post-capture cycle.

[0059] The combined mixed-mode / hydrophobic interaction flow-through (denoted as post-capture eluate) was continuously collected in a small surge vessel for further processing. The post-capture chromatography run was integrated with the preceding unit operation on day 23 and was run continuously for 47 days. Data summarizing the post-capture chromatography operational performance are shown in Figures 5A-5D.

[0060] A 0.1m effluent trap operated in tangential flow mode 2The virus was further processed in a Planova 20N (AK Bio) virus removal filter. The post-capture eluate was continuously transferred from the surge vessel to a pressurized loop containing a pressure vessel, a virus removal filter, a sterilizing-grade filter, and a peristaltic pump driving recirculation, as shown in Figure 6A. The pressure in the loop was automated such that the net volumetric flow rate through the virus filter (permeate flux) was equal to the net volumetric flow rate exiting the post-capture operation. The Planova 20N filter was replaced periodically based on a preliminary virus validation study. The virus-filtered material was continuously collected in a small surge vessel for further processing. The virus filtration operation was integrated with the preceding unit operation on day 29 and was run continuously for 41 days. Data summarizing the virus filtration operational performance are shown in Figures 6B-6F.

[0061] The virus-filtered material was then passed through a 0.065m 2 The mAb was further processed by ultrafiltration using an ILC single-pass TFF (SP-TFF) cassette and concentrated to a target mAb concentration of 65 g / L. The material was pumped from the post-virus filtration surge vessel to the inlet of the SP-TFF cassette at a flow rate that matched the outlet flow rate of the virus filtration operation. The retentate flow rate from the SP-TFF cassette was automatically controlled using a feedback loop with in-line measurement of product concentration to maintain the target concentration value. The concentrated mAb flow was then pumped directly to the inlet of a single-pass diafiltration (SP-DF) cassette for in-line buffer exchange. Diafiltration buffer was pumped to the buffer inlet of the SP-DF cassette and the buffer flow was automated to keep the ratio of buffer to product flow constant. The retentate flow rate was controlled to match the inlet flow rate. The buffer-exchanged DF retentate stream was then formulated by in-line addition of concentrated excipient solutions to produce the drug substance. The drug substance was further processed through a sterilizing grade filter to produce the drug substance, which was collected in a single-use container. Ultrafiltration and diafiltration The operation was integrated with the preceding unit operation on day 42 and run continuously for 29 days. The formulation operation and drug substance production were integrated on day 46 and run continuously for 25 days. Six batches of drug substance were produced during the 25 days. Data summarizing the ultrafiltration and diafiltration operational performance are shown in Figures 7A-7E and Figures 8A-8D. Figures 9A-9H show the properties of the drug substance produced. EXAMPLES

[0062] Continuous production of recombinant proteins Recombinant monoclonal antibodies (mAbs) were produced over a 27-day period using a 100 L intensive perfusion bioreactor coupled with dual ATFs. The cell-free mAb-containing medium (harvest) was continuously pumped into a 100 L harvest surge vessel for further processing.

[0063] Antibody-containing media was captured using two 1 L (8 x 20 cm) pre-packed and irradiated Protein A affinity chromatography columns in bind and elute MCC mode on a pilot-scale steam-in-place PCC skid. The capture step was run such that one of the two columns was always loaded from a harvest surge vessel and the average inlet flow rate of the capture step was matched to the average outlet flow rate of the perfusion bioreactor. The loading on the Protein A column was controlled by Delta UV to approximately 3% breakthrough. The eluates of the individual capture columns were collected in a 5 L mixing vessel prior to viral inactivation. The Protein A operation was integrated with the perfusion bioreactor on day 12 and run continuously for 15 days.

[0064] The individual Protein A eluates were mixed in a 5 L mixing vessel to ensure uniform flow for the viral inactivation unit operation. The outflow from the mixing vessel was automated so that the entire vessel was processed before the next Protein A elution cycle. The homogenized Protein A eluate was adjusted in-line to a target pH of 3.6 by adding 1 M acetic acid. The low pH adjusted Protein A eluate was then pumped through a gamma-irradiated 3D-printed tubular flow-through reactor (TFR) with a defined minimum residence time of at least 30 minutes to allow sufficient time at low pH to fully inactivate the virus. The viral inactivated Protein A eluate was then adjusted in-line to a target pH of 4.5 by adding 0.75 M sodium acetate. The adjusted viral inactivated Protein A eluate was then processed through a sterilizing grade filter and continuously collected in a small surge vessel for further processing. The viral inactivation operation was integrated with the preceding operation on day 13 and was run continuously for 14 days.

[0065] The conditioned virus-inactivated Protein A eluate was further processed in two orthogonal chromatography runs using mixed-mode anion exchange and hydrophobic interaction chromatography resins, operated in flow-through mode. The post-capture device consisted of two 0.2 L (5 × 10 cm) packed, gamma-irradiated mixed-mode columns and two 0.2 L (5 × 10 cm) packed, gamma-irradiated hydrophobic interaction columns, operated in MCC mode. The conditioned virus-inactivated Protein A eluate stream was continuously loaded onto one of the two mixed-mode columns, and the mixed-mode column flow-through was continuously loaded onto one of the two hydrophobic interaction columns. The mixed-mode and hydrophobic interaction columns were independently loaded to their respective loading capacities.

[0066] The post-capture chromatography run was integrated with the preceding unit operation on day 14 and was run for 13 consecutive days.

[0067] A 0.1m effluent trap operated in tangential flow mode 2The post-capture eluate was passed from the outlet of the hydrophobic interaction column through a pressure vessel, a viral removal filter, a sterilizing grade filter, and a recirculating filter. The virus was continuously transferred to a pressurized loop containing a peristaltic pump driving the ring. The pressure in the loop was automated such that the net volumetric flow rate through the virus filter (permeate flux) was equal to the net volumetric flow rate exiting the post-capture operation. The virus-filtered material was continuously collected in a small surge vessel for further processing. The virus filtration operation was integrated with the preceding unit operation on day 14 and was run continuously for 13 days.

[0068] The virus-filtered material is then placed in a 2 x 0.1 m 2 The virus filtered material was further processed by ultrafiltration using a gamma irradiated TFF capsule operated in single pass mode. The virus filtered material was concentrated to a target mAb concentration of 110 g / L. The material was delivered from the post virus filtration surge vessel to the inlet of the TFF capsule at a flow rate that matched the outlet flow rate of the virus filtration operation. The retentate flow rate from the TFF capsule was automatically controlled using a feedback loop with in-line measurement of product concentration to maintain the target concentration value. The concentrated mAb flow was then delivered directly to the inlet of a gamma irradiated single pass diafiltration (SP-DF) cassette for in-line buffer exchange. Diafiltration buffer was delivered to the buffer inlet of the SP-DF cassette and the buffer flow was automated to keep the ratio of buffer to product flow constant. The retentate flow rate was controlled to match the inlet flow rate. The buffer exchanged DF retentate stream was then formulated by in-line addition of concentrated excipient solutions to produce the drug substance. The addition of formulation buffer was controlled by a feedback loop and an in-line sensor measuring the level of excipients in the formulation stream. The drug substance was further processed through sterilizing grade filters to produce drug substance, which was collected in single-use containers. Ultrafiltration and diafiltration operations were integrated with the preceding unit operations on day 18, and formulation operations were integrated on day 20. Due to issues with in-line sensors, UF / DF and formulation operations did not reach steady state before the end of the campaign.

[0069] While the present disclosure has been described in terms of various embodiments, it is understood that variations and modifications will occur to those skilled in the art. It is therefore intended by the appended claims to cover all such equivalent variations that fall within the scope of the present disclosure as claimed. Moreover, the section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0070] Each embodiment described herein may be combined with any other embodiment unless expressly indicated to the contrary. In particular, any feature or embodiment indicated as being preferred or advantageous may be combined with any other feature or embodiment indicated as being preferred or advantageous unless expressly indicated to the contrary.

[0071] All documents cited in this application are expressly incorporated herein by reference.

Claims

1. 1. A method for continuously producing a recombinant protein, comprising: (a) capturing recombinant protein from a substantially cell-free sample using one or more capture chromatography systems and eluting the recombinant protein from the one or more capture chromatography systems to generate an eluate comprising the recombinant protein, wherein the eluate is homogenized into a single mixture comprising the recombinant protein; (b) subjecting the homogenized single mixture to viral inactivation; (c) subjecting the single homogenized mixture of step (b) to one or more post-capture chromatography systems and collecting the product output comprising the recombinant protein; (d) subjecting the product output of step (c) to viral filtration; (e) subjecting the second product output of step (d) to ultrafiltration and diafiltration to purify the recombinant protein; wherein steps (a) to (e) are carried out integrally and continuously.

2. 2. The method of claim 1, wherein virus inactivation comprises inactivation by a solvent-detergent solution, by heat inactivation, or by acidic pH inactivation.

3. Inactivation by acidic pH: (a) adding one or more solutions to adjust the pH of the homogenized single mixture in-line to a low pH; (b) incubating the prepared homogenized single mixture at low pH during which it becomes a virus inactivation mixture; The method of claim 2 comprising:

4. 4. The method of claim 3, wherein the period is between 15 minutes and 2 hours.

5. The substantially cell-free sample is removed from a perfusion bioreactor comprising host cells producing the recombinant protein, a fed-batch bioreactor comprising host cells producing the recombinant protein, or a clarified liquid culture comprising host cells producing the recombinant protein; The method of claim 1.

6. The method of claim 1 , wherein the one or more capture chromatography systems comprises a chromatography medium comprising resin beads, a porous membrane, or nanofibers.

7. 7. The method of claim 6, wherein the chromatographic medium is functionalized for affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, or mixed mode chromatography.

8. The method of claim 7, wherein the affinity chromatography medium is a Protein A-based resin.

9. 2. The method of claim 1, wherein the one or more capture chromatography systems is a cyclic countercurrent chromatography system (PCCS).

10. 4. The method of claim 3, wherein incubating the conditioned homogenized single mixture at low pH for a period of time resulting in viral inactivation is performed in a tubular flow reactor.

11. 4. The method of claim 3, wherein the addition of one or more solutions is based on an in-line recombinant protein concentration measurement and an in-line flow rate measurement.

12. 10. The method of claim 1, wherein the viral filtration is performed in a pressurized loop.

13. 13. The method of claim 12, wherein the pressurized loop comprises a pressure vessel, a viral removal filter, and a sterile filter.

14. 14. The method of claim 13, wherein the pressure vessel is a single-use, sealable, and sterilizable vessel.

15. 14. The method of claim 13, wherein the virus removal filter is an ultrafiltration membrane or a nanofilter.

16. 2. The method of claim 1, wherein the viral filtration is performed using dead-end filtration.

17. 17. The method of claim 16, wherein the dead-end filtration is performed using an ultrafiltration membrane or a nanofilter.

18. The method of claim 1 , wherein the ultrafiltration is performed by single-pass tangential flow filtration.

19. 10. The method of claim 1, wherein the diafiltration is performed using a single-pass diafiltration cassette.

20. The method of claim 1 , wherein the recombinant protein is an antibody or an antigen-binding fragment thereof.

21. 10. The method of claim 1, further comprising adding one or more excipients after step (e) to produce a therapeutic drug substance.

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