Continuous production of recombinant proteins

Abstract

This disclosure relates to methods and systems for the continuous production of recombinant proteins. In certain embodiments, this document relates to methods and systems for the continuous production of recombinant proteins using capture chromatography, post-capture chromatography, viral filtration, and ultrafiltration / dialysis filtration.

Description

Technical Field

[0001] This disclosure relates to methods and systems for the continuous production of recombinant proteins. In specific embodiments, this disclosure relates to methods and systems for the continuous production of recombinant proteins using capture chromatography, post-capture chromatography, viral filtration, and ultrafiltration / dialysis filtration. Prior Technology

[0002] Continuous manufacturing is a routine production method used in industries such as petrochemicals and food production. The benefits of continuous processes include steady-state operation, reduced equipment size, high volumetric productivity, simplified process flow, low cycle time, and reduced capital costs. Konstantinov et al., J. Pharm. Sci. 104(3): 813-20 (2015).

[0003] Continuous manufacturing is not yet widely implemented in the biopharmaceutical industry. Due to the significant differences in technological requirements between upstream and downstream development, experience in continuously operating downstream processes is limited. Therefore, the biopharmaceutical industry still needs integrated, continuous biomanufacturing methods that can operate for extended periods and minimize operator interaction. Summary of the Invention

[0004] This document provides a continuous method for producing recombinant proteins, the method comprising: (a) capturing the recombinant protein from a substantially cell-free sample using one or more capture chromatography systems, and flushing the recombinant protein from the one or more capture chromatography systems to produce an extract containing the recombinant protein, wherein one or more extracts are homogenized into a single mixture containing the recombinant protein; (b) subjecting the single mixture to one or more post-capture chromatography systems and collecting a product output containing the recombinant protein; and (c) subjecting the product output to ultrafiltration and dialysis filtration to purify the recombinant protein, wherein the method is integrated and continuous from step (a) to step (c).

[0005] This document also provides a manufacturing system for producing recombinant proteins, the manufacturing system comprising: (a) a first unit operation including a bioreactor containing host cells for producing 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 dialysis filtration system.

[0006] This document also provides a continuous method for producing recombinant proteins, the method comprising: (a) capturing the recombinant protein from a substantially cell-free sample using one or more capture chromatography systems and flushing the recombinant protein from the one or more capture chromatography systems to produce an extract containing the recombinant protein, wherein the extract is homogenized into a single mixture containing the recombinant protein; (b) subjecting the homogenized single mixture to viral inactivation; (c) subjecting the homogenized single mixture from step (b) to one or more post-capture chromatography systems and collecting a product output containing the recombinant protein; (d) subjecting the product output from the step to viral filtration; and (e) subjecting the product output from step (d) to ultrafiltration and dialysis filtration to purify the recombinant protein, wherein the method is integrated and continuous from step (a) to step (e).

[0007] Other aspects, embodiments, and implementations thereof will become apparent from the following detailed description and claims, and, where appropriate, from the accompanying drawings. Simple Explanation of the Diagram

[0008] Figure 1 is a schematic diagram illustrating the process flow of the method disclosed herein. The process flow includes a continuous process for producing recombinant proteins using online buffer, comprising: (1) producing recombinant proteins (labeled "BRX") in a bioreactor coupled with a dual alternating tangential flow (ATF) filter and cell separation; (2) capturing the recombinant proteins using one or more capture chromatography systems in a culture medium obtained in a bioreactor connected to the capture chromatography system (labeled "capture"); (3) viral inactivation of the culture medium (labeled "VI"); (4) passing the culture medium through one or more post-capture chromatography systems (labeled "post-capture post"); (5) viral filtration of the culture medium; and (6) ultrafiltration / dialysis filtration of the culture medium.

[0009] Figures 2A and 2B provide an overview of the unit operation integration of the methods and systems disclosed herein. Figure 2A shows the unit operation integration duration for sequential operations of each unit. Figure 2B shows the average protein residence time for each unit operation. The methods and systems disclosed herein produce approximately 5 kg of drug substance over a 25-day period.

[0010] Figures 3A to 3F are graphs illustrating the operational performance of the method and system disclosed herein for protein A. Figure 3A shows the concentration of protein A extract (g / L), Figure 3B shows the host cell protein residue in the protein A extract, Figure 3C shows the residual protein in the protein A extract, Figure 3D shows the inlet mass flow rate of protein A, and Figures 3E and 3F show the ultraviolet chromatography patterns of protein A.

[0011] Figures 4A to 4G are graphs illustrating the viral inactivation 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 alkali 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.

[0012] Figures 5A to 5D are graphs illustrating the post-capture performance of the method and system disclosed herein. Figure 5A shows residual protein A in the post-capture extract, Figure 5B shows the protein concentration in the post-capture extract, Figure 5C shows a UV chromatography image, and Figure 5D shows residual host cell proteins in the post-capture extract.

[0013] Figure 6A shows a schematic diagram of virus filtration using the method and system disclosed herein. Figures 6B to 6G are graphs illustrating the operational performance of the virus filtration using the method and system disclosed herein. Figure 6B shows the protein concentration after virus filtration, Figure 6C shows the high molecular weight species after virus filtration, Figure 6D shows the protein mass flux during virus filtration, Figure 6E shows the filtrate volume flux during virus filtration, Figure 6F shows the transmembrane pressure during virus filtration, and Figure 6G shows the filter performance during virus filtration.

[0014] Figures 7A to 7E are graphs illustrating the ultrafiltration 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 volume flux during ultrafiltration, and Figure 7E shows transmembrane pressure during ultrafiltration.

[0015] Figures 8A to 8E are graphs illustrating the dialysis filtration performance of the methods and systems disclosed herein. Figure 8A shows the permeate volume flux during dialysis filtration, Figure 8B shows the protein mass flux during dialysis filtration, Figure 8C shows the transmembrane pressure during dialysis filtration, Figure 8D shows the dialysis filtration ratio, and Figure 8E shows the filter performance during dialysis filtration.

[0016] Figures 9A to 9H are graphs showing the properties of pharmaceutical substances produced using the methods and systems disclosed herein. Figure 9A shows the concentration of the pharmaceutical substance, Figure 9B shows the high molecular weight species of the pharmaceutical substance, Figure 9C shows the charge characteristic curve of the pharmaceutical substance, Figure 9D shows the gravimetric osmotic concentration of the pharmaceutical substance, Figure 9E shows the pH of the pharmaceutical substance, Figure 9F shows the host cell protein content of the pharmaceutical substance measured by ELISA, Figure 9G shows the non-reducing purity of the pharmaceutical substance, and Figure 9H shows the residual protein A of the pharmaceutical substance. Implementation

[0017] This disclosure relates to methods and systems for the continuous production of recombinant proteins. In specific embodiments, this disclosure relates to methods and systems for producing recombinant proteins using capture chromatography, post-capture chromatography, and ultrafiltration / dialysis filtration. The methods and systems described herein provide continuous and time-efficient production of recombinant proteins.

[0018] As used 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 the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms.

[0019] In some embodiments, this document provides a continuous method for producing recombinant proteins, the method comprising capturing the recombinant protein from a substantially cell-free sample using one or more capture chromatography systems, flushing the recombinant protein from the one or more capture chromatography systems to produce a flush containing the recombinant protein, wherein the flush is homogenized into a single mixture containing the recombinant protein, the homogenized single mixture is passed through one or more post-capture chromatography systems and a product output containing the recombinant protein is collected, and the product output is subjected to ultrafiltration and dialysis filtration to purify the recombinant protein, wherein the method is integrated and operated continuously.

[0020] This document also provides a manufacturing system for producing recombinant proteins, the manufacturing system comprising: a first operating unit including a bioreactor containing host cells for producing the recombinant protein; a second operating unit including one or more capture chromatography systems; a third operating unit including one or more post-capture chromatography systems; and a fourth operating unit including an ultrafiltration system and a dialysis filtration system.

[0021] In some embodiments, the manufacturing system includes a fifth operating unit located between a first operating unit and a second operating unit, wherein the fifth operating unit includes a subsystem for performing virus inactivation. In some embodiments, the manufacturing system includes a sixth operating unit located between a third operating unit and a fourth operating unit, wherein the sixth operating unit includes a second subsystem for performing virus filtration. In some embodiments, the manufacturing system includes a seventh unit operation, wherein the seventh unit operation includes a third subsystem, wherein the third subsystem includes an in-line excipient for formulating therapeutic pharmaceutical substances.

[0022] As used herein, “substantially cell-free” means a sample that is at least or about 90% free of (e.g., at least or about 95%, 96%, 97%, 98%, or at least or about 99%, or about 100%) specified substances (such as mammalian cells).

[0023] In certain embodiments, the substantially cell-free sample is taken from a perfusion bioreactor containing host cells that produce the recombinant protein, a fed-batch bioreactor containing host cells that produce the recombinant protein, or a clear liquid nutrient solution containing host cells that produce the recombinant protein.

[0024] As used herein, "liquid culture medium" refers to a fluid containing sufficient nutrients to allow cells to grow or proliferate in vitro. For example, a liquid culture medium may contain one or more of the following: amino acids (e.g., 20 amino acids), purines (e.g., hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate. In some embodiments, the liquid culture medium may contain serum from mammals. In some embodiments, the liquid culture medium does not contain serum or other extracts from mammals (defined as liquid culture medium). In some embodiments, the liquid culture medium may contain trace amounts of metals, mammalian growth hormones, and / or mammalian growth factors. Other suitable liquid culture media are known in the art and are commercially available.

[0025] As used herein, a "perfusion bioreactor" refers to a bioreactor containing a plurality of cells in a first liquid culture medium, wherein the culture of cells present in the bioreactor includes periodically or continuously removing the first liquid culture medium and simultaneously or shortly thereafter adding a substantially equal volume of a second liquid culture medium to the bioreactor. In some embodiments, during the culture period (e.g., at a daily culture medium refeed rate), the volume of the first liquid culture medium removed and added varies incrementally over increasing time periods (e.g., a period of approximately 24 hours, a period between approximately 1 minute and approximately 24 hours, or a period longer than 24 hours). The ratio of culture medium removed and replaced daily can vary depending on the specific cells being cultured, the initial inoculation density, and the cell density at a particular time.

[0026] As used in this article, a "batch-feed bioreactor" refers to a bioreactor containing multiple cells in which one or more feed streams are used to intermittently or continuously provide the essential nutrients for cell growth and product formation.

[0027] As used herein, “clarified liquid culture medium” refers to a liquid culture medium obtained from bacterial or yeast cell cultures that is substantially free of (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% free of) bacterial or yeast cells.

[0028] As used in this article, "recombinant protein" refers to immunoglobulins, protein fragments, engineered proteins, blood factors, nanoantibodies, or enzymes, including antibodies or antibody fragments thereof.

[0029] As used herein, "unit operation" refers to a functional step that can be performed in a method for manufacturing recombinant proteins, or a component of a system used in the process of manufacturing recombinant proteins. For example, an operation unit may be filtration (e.g., removing contaminating bacteria, yeast viruses, or mycobacteria, and / or specific substances from a fluid containing recombinant therapeutic proteins), capture, removal of epitope tags, purification, retention or storage, refining, virus inactivation, adjustment of ion concentration and / or pH of a fluid containing recombinant proteins, and removal of unwanted salts.

[0030] As used herein, the term "continuous method" or "continuous system" refers to a method or system in which fluid is continuously supplied through at least a portion of unit operations. A unit operation is continuous if it can handle a continuous flow input over a long period. Continuous unit operations have a minimal internal holding volume. The output can be continuous or discretized in small packets generated in a loop. A method is continuous if it consists of integrated (physically connected) continuous unit operations with zero or minimal holding volumes between them.

[0031] As used herein, an "integrative approach" refers to a method that uses structural elements that work together to achieve a specific result (e.g., the production of recombinant proteins from liquid culture media).

[0032] As used in this article, "extract" refers to the fluid discharged from a chromatography column or membrane that contains detectable amounts of recombinant protein.

[0033] As used in this article, "chromatographic medium" refers to the material packed into a chromatography column.

[0034] In some embodiments, the systems disclosed herein are closed systems. Closed systems include unit operations designed and operated to limit exposure to the external environment. Materials may be introduced into a closed system, but must be added in a manner that avoids product exposure to the indoor environment.

[0035] The methods and systems disclosed herein include using one or more capture chromatography systems to capture recombinant proteins and flushing the recombinant proteins from the one or more capture chromatography systems to produce a flush containing the recombinant proteins. The chromatography media contained in the one or more capture chromatography systems may be resins utilizing capture mechanisms (e.g., protein A binding capture mechanism, protein G binding capture mechanism, antibody or antibody fragment binding capture mechanism, substrate binding capture mechanism, cofactor binding capture mechanism, tag binding capture mechanism, and / or aptamer binding capture mechanism). In some embodiments, the one or more capture chromatography systems comprise chromatography media composed of resin particles, porous membranes, or nanofibers. In some embodiments, the chromatography media are functionalized for affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, or mixed-mode chromatography. In some embodiments, the affinity chromatography media is a protein A-based resin.

[0036] In some embodiments, the one or more capture chromatography systems are periodic countercurrent chromatography systems (PCCS). In some embodiments, the PCCS includes two chromatography columns or includes a modified ÄKTA system (GE Healthcare, Piscataway, New Jersey) capable of operating with up to, for example, four, five, six, seven, eight, or more than eight columns. In some embodiments, the PCCS utilizes a column switching mechanism. Column switching events can be triggered by detecting the level of the recombinant protein (by UV absorbance corresponding to a certain level of the recombinant protein in the fluid passing through the chromatography system), a specific volume of liquid (e.g., buffer), or a specific elapsed time.

[0037] In order to capture recombinant proteins using a capture chromatography system, the chromatography steps of loading, washing, rinsing, and regenerating the capture chromatography system must be performed in sequence.

[0038] In some embodiments, the extract 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 passed through one or more post-capture chromatography systems, and the product output containing the recombinant protein is collected. The post-capture chromatography system is used to remove residual trace or small amounts of contaminants or impurities from the fluid containing the recombinant protein at near-desired final purity. In some embodiments, the one or more post-capture chromatography systems comprise a chromatography medium composed of resin particles, 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 periodic countercurrent chromatography systems (PCCS).

[0039] In order to perform post-capture processing of recombinant proteins using a post-capture chromatography system, the chromatography steps of loading, washing, rinsing, and regenerating the post-capture chromatography system must be performed in sequence.

[0040] In some embodiments, the methods and systems include ultrafiltration (UF) and / or dialysis filtration (DF) to further purify and concentrate recombinant proteins. UF / DF can increase the concentration of recombinant proteins and replace buffer salts with specially formulated buffers. Ultrafiltration (UF) refers to a type of membrane filtration in which hydrostatic pressure forces liquid against a semipermeable membrane. In some embodiments, UF is performed using tangential flow filtration (TFF), which includes single-pass TFF and high-performance tangential flow filtration (HPTFF). Single-pass tangential flow filtration (SPTFF) refers to any TFF system in which the conversion rate (permeate flow rate divided by inlet feed flow rate) of a single pass through the module is sufficiently high that the system can operate without a recovery (recirculation) loop for permeate. SPTFF can be used for in-line concentration between other unit operations, for example to reduce the processing volume prior to chromatography or precipitation steps. Zydney, Biotechnol. Bioeng. 113(3): 465-75 (2016).

[0041] As used herein, "dialysis filtration" refers to a method of removing, displacing, or reducing the concentration of salts or buffer components from a protein solution containing proteins such as antibodies, peptides, nucleic acids, and other biomolecules using an ultrafiltration membrane. Continuous dialysis filtration (also known as constant-volume dialysis filtration) involves washing out the original buffer salts (or other low-molecular-weight species) from the osmotic residue by adding water or a fresh buffer solution (such as a formulation buffer) to form a formulation containing recombinant peptides. Typically, the fresh buffer solution is added at the same rate as the filtrate is produced, so that the volume of the osmotic residue and the concentration of the product do not change significantly during dialysis filtration. In certain embodiments, the dialysis filtration is performed using a single-pass dialysis filter cartridge.

[0042] In some embodiments, the homogenized single mixture is subjected to virus inactivation before being subjected to one or more post-capture chromatography systems. In some embodiments, virus inactivation includes solvent-detergent solution inactivation, heat inactivation, or acidic pH inactivation.

[0043] For solvent-detergent virus inactivation, the steps involve subjecting a sample containing recombinant proteins to an organic solvent and a detergent. The solvent-detergent combination can be any solvent-detergent combination known in the art, such as tributyl phosphate and Triton X-100™, Tween 80™ and sodium cholate, and others.

[0044] Virus inactivation by heat involves subjecting a sample containing recombinant proteins to a high temperature. In some embodiments, the method includes heating the sample to a temperature greater than or equal to 45ºC, 46ºC, 47ºC, 48ºC, and up to approximately greater than or equal to 49ºC, 50ºC, 51ºC, and higher. In some embodiments, the sample is heated to a temperature between 45ºC and 65ºC.

[0045] The heating time for the sample can vary. For example, in some embodiments, the sample is heated to the target temperature for a duration between 1 minute and 6 hours. In some embodiments, the sample is heated to the target temperature for a duration between 10 and 180 minutes, between 20 and 180 minutes, between 20 and 60 minutes, or between 20 and 40 minutes.

[0046] In some embodiments, the acidic pH inactivation system is performed by adjusting a homogenized single mixture to a low pH using one or more solutions online, and culturing the adjusted homogenized single mixture at the low pH for a period of time to produce a virus-inactivated mixture.

[0047] The methods and systems disclosed herein include online monitoring, including the level of recombinant protein detection (detected by UV absorbance), flow rate detection, and / or the volume of detection fluid (e.g., buffer). Monitoring of recombinant protein concentration (e.g., via UV monitoring) can be determined using any tool with feedback control and capable of online measurement of product concentration.

[0048] In some embodiments, the homogenized single mixture system is exposed to a low pH for 15 minutes to 2 hours. In a particular embodiment, the low pH is between 3 and 5. The choice of pH level depends on the stability profiles of the recombinant protein and other buffer components. After viral 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 the method.

[0049] In some embodiments, virus inactivation is carried out in a tubular flow reactor, as described by 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.

[0050] In some embodiments, a virus removal step, such as virus filtration, is included after collecting 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 viral inactivation treatment. In some embodiments, virus filtration is performed in a pressurized loop, wherein the pressurized loop includes a pressure vessel, a virus removal filter, and a sterilizing filter. In some embodiments, the virus removal filter in the pressurized loop is a hollow fiber virus filter. In some embodiments, the pressure vessel is a single-use, closed, and sterilizable vessel. As used herein, the term "sterilizable" refers to a vessel formed of a material compatible with known sterilization methods. In some embodiments, virus filtration is performed using a closed-end filtration system. As used herein, "closed-end filtration" refers to a filtration in which the entire fluid stream being filtered passes through the filter without any recovery or effluent flow. In some embodiments, the virus removal filter is an ultrafilter or a nanofilter. Examples of suitable virus filtration systems are disclosed in International Publication No. WO 2018 / 035116, which is incorporated herein by reference.

[0051] In some embodiments, the first unit operation includes an outlet connected to an inlet on the second unit operation, wherein the second unit operation includes an outlet connected to an inlet on the third unit operation, and wherein the third unit operation includes an outlet connected to an inlet on the fourth unit operation. In some embodiments, the first unit operation includes an outlet connected to an inlet on the fifth unit operation, wherein the fifth unit operation includes an outlet connected to an inlet on the second unit operation, wherein the second unit operation includes an outlet connected to an inlet on the third unit operation, wherein the third unit operation includes an outlet connected to an inlet on the sixth unit operation, and wherein the sixth unit operation includes an outlet connected to an inlet on the fourth unit operation.

[0052] In some embodiments, the system disclosed herein includes a buffer dish between each of the first unit operation, the second unit operation, the third unit operation, and the fourth unit operation. The buffer dish is capable of holding any liquid culture before it is moved to the next operating unit.

[0053] The system described herein may also include fluid conduits disposed between any unit operations. Suitable fluid conduits may be tubes made of polyethylene, polycarbonate, or plastic. The fluid conduits may also include one or more of the following in any combination: one or more online buffer conditioning reservoirs in fluid communication with the fluid conduits and positioned such that buffer stored in the one or more online buffer conditioning reservoirs is added to the fluid present in the fluid conduits; and one or more filters disposed in the fluid conduits such that they are capable of filtering (e.g., removing bacteria) the fluid present in the fluid conduits.

[0054] In some embodiments, the system provided herein includes a pump system. The pump system may 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.

[0055] In some embodiments, one or more excipients are added to the product output after ultrafiltration / dialysis to produce a therapeutic pharmaceutical substance.

[0056] As used herein, "therapeutic pharmaceutical substance" refers to a substance containing a recombinant protein that has been adequately purified or separated from contaminating proteins, lipids, and nucleic acids (e.g., contaminating proteins, lipids, and nucleic acids present in liquid culture media or from host cells (e.g., mammalian, yeast, or bacterial host cells) and biocontaminants (e.g., viral and bacterial contaminants) and can be formulated into a pharmaceutical preparation without any further substantial purification and / or cleansing steps.

[0057] In some embodiments, the system disclosed herein is mounted on a slide rail. As used herein, a "slide rail" refers to a three-dimensional solid structure that can serve as a platform or support for the system described herein. If the slide rail includes one or more movable structures (e.g., wheels, rollers, etc.), it can impart mobility to the system or a portion thereof.

[0058] In some embodiments, the method and system have a recombinant protein recovery rate of at least about 40%, 50%, 55%, or 60%, and at most about 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the purification recovery rate is at least about 60% to about 70%.

[0059] In some embodiments, the methods and systems described herein result in a net yield of recombinant protein in a therapeutic protein pharmaceutical substance of at least about 5 g / day, 10 g / day, or 20 g / day, 30 g / day, or 40 g / day, and at most about about 200 g / day, 300 g / day, 400 g / day, 500 g / day, or 1000 g / day, over a continuous period of at least about 5 days, 10 days, 20 days, or 30 days, and at most about about 280 days, 290 days, 300 days, 310 days, 320 days, 330 days, 340 days, 350 days, or 365 days. [Example] []

[0060] The following examples illustrate specific embodiments of this disclosure and their various uses. These examples are for illustrative purposes only and should not be construed as limiting the scope of this disclosure in any way. [] [Example] [1] Continuous production of recombinant proteins []

[0061] A 100-L enhanced perfusion bioreactor with dual ATFs was used to produce recombinant monoclonal antibodies (mAbs) over a 70-day period. Cell-free culture medium containing mAbs (harvest) was continuously pumped into 100 L harvest buffer dishes for further processing.

[0062] Antibody-containing culture media were captured using 2 x 1-L (8 x 20 cm) pre-packaged and irradiated protein A affinity chromatography columns, operated in bind-and-elute MCC mode on a pilot-scale steam-in-place (PCC) rail. The capture step was performed such that one of the two columns was always loaded from the harvest buffer dish, and the average inlet flow rate into the capture step was matched to the average outlet flow rate leaving the perfusion bioreactor. The load on the protein A column was controlled to approximately 3% breakthrough using Delta UV. The extracts from each capture column were collected in a 5-L mixing dish before virus inactivation. The protein A operating system was integrated into the perfusion bioreactor on day 12 and operated continuously for 58 days. Figures 3A to 3F show the summary of protein A operating performance data.

[0063] Each protein A extract system was mixed in a 5-L mixing vessel to ensure a homogeneous flow for virus inactivation unit operation. The effluent from the mixing vessel was automated, ensuring the entire vessel system was processed before the next protein A extract loop. The homogenized protein A extract system was adjusted online to its target pH of 3.6 by adding 1 M acetic acid. The low-pH-adjusted protein A extract was then pumped through a tubular flow reactor (TFR) with a minimum residence time of ≥ 30 min to ensure sufficient time for complete virus inactivation at the low pH. The virus-inactivated protein A extract system was then adjusted online to its target pH of 4.5 by adding 0.75 M sodium acetate. The adjusted virus-inactivated protein A extract was then processed through a sterile filter and continuously collected in small buffer vessels for further processing. The virus inactivation operation was integrated with the previous operation on day 15 and run continuously for 55 days. Figures 4A to 4G show the summary data for the virus inactivation operation performance.

[0064] The adjusted viral-inactivated protein A effervescent was further processed by two orthogonal chromatography operations using a mixed-mode anion exchange resin and a hydrophobic interaction resin, operated in flow-through mode. For this example process, there was no inter-step adjustment between the two post-capture chromatography operations, allowing the flow material to be loaded directly from the mixed-mode column onto the hydrophobic interaction column. The post-capture train consisted of two 0.2 L (5 x 10 cm) pre-packed mixed-mode columns and two 0.2 L (5 x 10 cm) pre-packed hydrophobic interaction columns, operated in MCC mode on PCC rails, identical to the protein A operation. The adjusted and viral-inactivated protein A effervescent was continuously loaded onto one of the two parallel post-capture trains, with column loading automated, such that each post-capture loop contained a load equivalent to approximately twice the mass of the protein A effervescent.

[0065] The combined mixed-mode / hydrophobic interaction flow products (labeled post-capture extracts) were continuously collected in small buffer dishes for further processing. The post-capture chromatography operation was integrated with the previous unit operation on day 23 and run continuously for 47 days. Figures 5A-5D show the summary data for the performance of the post-capture chromatography operation.

[0066] The post-capture extract was further processed through a 0.1 m² Planova 20N (AK Bio) virus removal filter, operating in tangential flow mode. The post-capture extract was continuously transferred from the buffer vessel to a pressurized loop containing a pressure vessel, a virus removal filter, a sterile filter, and a peristaltic pump to drive recirculation, as shown in Figure 6A. The pressure in the loop was automated, so the net volumetric flow rate (permeate flux) through the virus filter was equal to the net volumetric flow rate exiting the post-capture operation. Based on preliminary virus validation studies, the Planova 20N filter was periodically replaced. The virus-filtered material was continuously collected in a small buffer vessel for further processing. On day 29, the virus filtration operation was integrated with the previous unit operation and ran continuously for 41 days. Figures 6B to 6F show the summary data for the virus filtration operation performance.

[0067] The virus-filtered material was further processed by ultrafiltration using a 0.065 m² ILC single-pass TFF (SP-TFF) cartridge and concentrated to a target mAb concentration of 65 g / L. The material was pumped from the post-virus filter buffer dish to the inlet of the SP-TFF cartridge at a flow rate matched to the outlet flow rate exiting the virus filtration operation. A feedback loop was used to automatically control the osmotic flow rate exiting the SP-TFF cartridge by measuring the product concentration online, maintaining the target concentration. The concentrated mAb stream was then pumped directly to the inlet of a single-pass dialysis filter (SP-DF) cartridge for online buffer exchange. Dialysis filter buffer was pumped to the buffer inlet of the SP-DF cartridge, and the buffer flow rate was automated to maintain a constant buffer-to-product flow rate ratio. The osmotic flow rate was controlled to match the inlet flow rate. The buffer-exchanged DF osmotic flow was then formulated by adding concentrated excipient solution online to produce a formulated material. The formulated material was further processed through a sterile filter to produce the drug substance, which was collected in single-use containers. On day 42, ultrafiltration and dialysis were integrated with the previous unit operations and run continuously for 29 days. On day 46, the preparation and drug production were integrated and run continuously for 25 days. During the 25-day period, six batches of drug were produced. Figures 7A-7E and 8A-8D summarize the performance data of the ultrafiltration and dialysis operations. Figures 9A-9H show the properties of the produced drug. [] [Example] [2] Continuous production of recombinant proteins []

[0068] A 100-L enhanced perfusion bioreactor with dual ATFs was used to produce recombinant monoclonal antibodies (mAbs) over a 27-day process. Cell-free culture medium containing mAb (harvest) was continuously pumped into 100 L harvest buffer dishes for further processing.

[0069] Antibody-containing culture media were captured using 2 x 1-L (8 x 20 cm) pre-packaged and irradiated protein A affinity chromatography columns, operated in binding and extraction MCC mode on a lead-scale steam-in-place PCC slide. The capture step was performed such that one of the two columns was always loaded from the harvest buffer dish, and the average inlet volumetric flow rate entering the capture step was matched to the average outlet volumetric flow rate leaving the perfusion bioreactor. The loading on the protein A column was controlled to approximately 3% breakthrough using Delta UV. The extracts from each capture column were collected in 5-L mixing dishes before viral inactivation. The protein A operation was integrated into the perfusion bioreactor on day 12 and operated continuously for 15 days.

[0070] The respective protein A extract systems were mixed in 5-L mixing vessels to ensure a homogeneous flow for virus inactivation unit operations. The effluent from the mixing vessels was automated, ensuring the entire vessel system was processed before the next protein A extract loop. The homogenized protein A extract system was adjusted online to its target pH of 3.6 by adding 1 M acetic acid. The low-pH-adjusted protein A extract system was then pumped through a 3D-printed and gamma-ray-irradiated tubular flow reactor (TFR) with a defined minimum residence time ≥ 30 min to ensure sufficient time for complete virus inactivation at the low pH. The virus-inactivated protein A extract system was then adjusted online to its target pH of 4.5 by adding 0.75 M sodium acetate. The adjusted virus-inactivated protein A extract system was then processed through a sterile filter and continuously collected in small buffer vessels for further processing. The virus inactivation operation was integrated with the previous operation on day 13 and run continuously for 14 days.

[0071] The adjusted viral-inactivated protein A effervescent stream was further processed by two orthogonal chromatography operations using a mixed-mode anion exchange resin and a hydrophobic interaction resin, in flow-through mode. The post-capture column consisted of a 2 x 0.2 L (5 x 10 cm) pre-packed and gamma-ray-irradiated mixed-mode column operating in MCC mode and a 2 x 0.2 L (5 x 10 cm) pre-packed and gamma-ray-irradiated hydrophobic interaction column. The adjusted and viral-inactivated protein A effervescent stream was continuously loaded onto one of the two mixed-mode columns, and the flow-through from the mixed-mode column was continuously loaded onto one of the two hydrophobic interaction columns. The mixed-mode column and the hydrophobic interaction column were loaded to their respective loading capacities.

[0072] On day 14, the post-capture chromatography operation was integrated with the previous unit operation and run continuously for 13 days.

[0073] The captured extract was further processed through a 0.1 m² Planova 20N (AK Bio) virus removal filter, operated in tangential flow mode. The captured extract was continuously transferred from the outlet of the hydrophobic interaction column to a pressurized loop containing a pressure vessel, a virus removal filter, a sterile filter, and a peristaltic pump to drive recirculation. The pressure in the loop was automated, so the net volumetric flow rate (permeate flux) through the virus filter was equal to the net volumetric flow rate from the post-capture operation. The filtered material was continuously collected in a small buffer vessel for further processing. The virus filtration operation was integrated with the previous unit operation on day 14 and ran continuously for 13 days.

[0074] The virus-filtered material was further processed by ultrafiltration using tandem 2 x 0.1 m² gamma-ray irradiated TFF capsules, operated in single-pass mode. The virus-filtered material was concentrated to a target mAb concentration of 110 g / L. The material was pumped from the post-virus filter buffer dish to the inlet of the TFF capsule at a flow rate matched to the outlet flow rate leaving the virus filtration operation. A feedback loop was used to automatically control the filtrate flow rate leaving the TFF capsule by measuring the product concentration online to maintain the target concentration. The concentrated mAb flow was then pumped directly to the inlet of a gamma-ray irradiated single-pass dialysis filter (SP-DF) cartridge for online buffer exchange. Dialysis filter buffer was pumped to the buffer inlet of the SP-DF cartridge, and the buffer flow rate was automated, maintaining a constant buffer-to-product flow rate. The filtrate flow rate was controlled to match the inlet flow rate. The buffer-exchanged DF filtrate flow was then formulated by adding a concentrated excipient solution online to produce a formulated material. The addition of prepared buffer solution was controlled via a feedback loop and an online sensor (which measures the excipient level in the prepared stream). The prepared material was further processed through a sterile filter to produce the drug substance, which was collected in single-use vessels. Ultrafiltration and dialysis filtration were integrated with previous unit operations on day 18, and the preparation operation was integrated on day 20. Due to issues with the online sensor, the UF / DF and preparation operations did not reach a stable state before the end of the campaign.

[0075] While this document has described various embodiments, it should be understood that variations and modifications will occur to those skilled in the art. Therefore, the appended claims are intended to cover all such equivalent variations within the scope claimed herein. Furthermore, the section headings used herein are for architectural purposes only and are not to be construed as limiting the subject matter.

[0076] Unless expressly indicated to the contrary, each embodiment described herein may be combined with any other one or more embodiments. In particular, unless expressly indicated to the contrary, any feature or embodiment indicated as preferred or advantageous may be combined with any other one or more features or embodiments indicated as preferred or advantageous.

[0077] All references cited in this application are explicitly incorporated into the content disclosed herein through citation.

[0078] none

Claims

1. A continuous method for producing recombinant proteins, the method comprising: (a) The recombinant protein is captured from a substantially cell-free sample using one or more capture chromatography systems, and the recombinant protein is flushed from the one or more capture chromatography systems to produce a flush containing the recombinant protein, wherein the flush is homogenized into a single mixture containing the recombinant protein; (b) The homogenized single mixture of step (a) is subjected to viral inactivation; (c) The homogenized single mixture of step (b) is passed through one or more post-capture chromatography systems, and a product output containing the recombinant protein is collected; (d) The product output of step (c) is subjected to viral filtration; and (e) The product output of step (d) is subjected to ultrafiltration and dialysis filtration to purify the recombinant protein; wherein each step is performed by unit operations, each unit operation from step (a) to step (e) being physically connected, and the connected unit operations from step (a) to step (e) process a continuous stream from step (a) to step (e), the continuous stream containing the recombinant protein from step (a) to step (e).

2. The method as described in claim 1, wherein virus inactivation includes solvent-detergent solution inactivation, heat inactivation, or acidic pH inactivation.

3. The method as described in claim 2, wherein the acidic pH deactivation comprises: (i) Adjust the homogenized single mixture of step (b) to a low pH by adding one or more solutions; (ii) The adjusted homogenized single mixture from step (i) is incubated at the low pH for a period of time to produce virus inactivation.

4. The method as described in claim 3, wherein the time is between 15 minutes and 2 hours.

5. The method of claim 1, wherein the substantially cell-free sample is taken from a perfusion bioreactor containing host cells that produce the recombinant protein, a fed-batch bioreactor containing host cells that produce the recombinant protein, or a clear liquid culture containing host cells that produce the recombinant protein.

6. The method of claim 1, wherein the one or more capture chromatography systems and the one or more post-capture chromatography systems comprise chromatography media comprising resin particles, porous membranes, or nanofibers.

7. The method of claim 6, wherein the chromatography medium is functionalized for use in 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. The method of claim 1, wherein the one or more capture chromatography systems are periodic countercurrent chromatography systems (PCCS).

10. The method of claim 3, wherein the conditioning and homogenization of the single mixture in step (i) is incubated at the low pH for a period of time to produce virus inactivation in a tubular flow reactor.

11. The method as described in claim 3, wherein the addition of one or more solutions is based on online recombinant protein concentration measurement and online flow rate measurement.

12. The method as described in claim 1, wherein the virus filtering is performed in a pressurized loop.

13. The method of claim 12, wherein the pressurization circuit includes a pressure vessel, a virus removal filter, and a sterilization filter.

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

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

16. The method of claim 1, wherein the virus filtering is performed using closed-end filtering.

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

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

19. The method of claim 1, wherein the dialysis filtration is performed using a single-pass dialysis filtration.

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

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

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