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 on April 3, 2019, the disclosure of which is hereby expressly incorporated by reference herein.

[0002] This disclosure relates to methods and systems for continuously producing recombinant proteins. In certain embodiments, this disclosure relates to methods and systems that use capture chromatography, post - capture chromatography, virus filtration, and ultrafiltration / diafiltration for continuously producing recombinant proteins.

Background Art

[0003] Continuous manufacturing is a production method that is routinely carried out in industries such as petrochemicals and food production. The advantages of continuous processes include steady operation, downsizing of equipment, high volumetric productivity, rationalization of process flow, low cycle time, and reduction of capital costs. Non - Patent Document 1.

Prior Art Documents

Non - Patent Documents

[0004]

Non - Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the biopharmaceutical industry, continuous manufacturing is not widely implemented. Since the technical needs in upstream development and downstream development are very different, the experience of continuous operation downstream is limited. Therefore, in the biopharmaceutical industry, an integrated continuous bioprocess that can operate for a long time with minimal operator intervention is needed. [Means for solving the problem]

[0006] A method for the continuous production of recombinant proteins is provided herein, comprising: (a) capturing recombinant proteins from a substantially cell-free sample using one or more capture chromatography systems, eluting the recombinant proteins from one or more capture chromatography systems to produce elutes containing recombinant proteins, wherein one or more elutes are homogenized into a single mixture containing recombinant proteins; (b) subjecting the single mixture to one or more post-capture chromatography systems to collect the output of a product containing recombinant proteins; and (c) subjecting the output of the product to ultrafiltration and diafiltration to purify the recombinant proteins, wherein the method is provided herein in which steps (a) through (c) are carried out integrally and continuously.

[0007] Furthermore, the present invention provides a manufacturing system for producing recombinant proteins, comprising: (a) a first unit operation comprising a bioreactor comprising host cells that produce recombinant proteins; (b) a second unit operation comprising one or more capture chromatography systems; (c) a third unit operation comprising one or more post-capture chromatography systems; and (d) a fourth unit operation comprising an ultrafiltration system and a diafiltration system.

[0008] Furthermore, a method for continuously producing recombinant proteins is provided herein, comprising: (a) capturing recombinant proteins from a substantially cell-free sample using one or more capture chromatography systems, eluting the recombinant proteins from one or more capture chromatography systems to produce an elute containing recombinant proteins, wherein the elute is homogenized into a single mixture containing recombinant proteins; (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 to collect the output of a product containing recombinant proteins; (d) subjecting the output of the product from step (c) to viral filtration; and (e) subjecting the output of the product from step (d) to ultrafiltration and diafiltration to purify the recombinant proteins, wherein the method is carried out integrally and continuously from step (a) to step (e).

[0009] Other aspects, embodiments, and configurations will become apparent from the following detailed description and claims, with reference to the accompanying drawings as appropriate. [Brief explanation of the drawing]

[0010] [Figure 1] This is a schematic diagram showing the process flow of the method disclosed herein. The process flow includes a continuous in-line buffer-based process for recombinant protein production, comprising: (1) producing recombinant protein in a bioreactor coupled with a dual alternating tangential flow (ATF) filter and cell separation (referred to as "BRX"), (2) capturing recombinant protein using one or more capture chromatography systems to capture recombinant protein present in the culture medium obtained from the bioreactor connected to the capture chromatography system (referred to as "capture"), (3) inactivating the virus in the medium (referred to as "VI"), (4) subjecting the medium to one or more post-capture chromatography systems (referred to as "post-capture"), (5) viral filtration of the medium, and (6) ultrafiltration / dialysis filtration of the medium. [Figure 2] Figures 2A and 2B show an overview of the integration of unit operations of the methods and systems disclosed herein. Figure 2A shows the duration of the unit operation integration for continuous operation of each unit operation. Figure 2B shows the average protein residence time for each unit operation. The methods and systems disclosed herein produced approximately 5 kg of pharmaceutical material in 25 days. [Figure 3-1] Figures 3A to 3F are graphs showing the operational performance of protein A in the methods and systems disclosed herein. Figure 3A shows the concentration (g / L) of protein A eluate, Figure 3B shows the residual host cell protein of protein A eluate, Figure 3C shows the residual protein of protein A eluate, Figure 3D shows the inlet mass flow rate of protein A, and Figures 3E and 3F show the ultraviolet chromatogram of protein A. [Figure 3-2] Continuation of Figure 3-1. [Figure 3-3] Continuation of Figure 3-2. [Figure 4-1] Figures 4A to 4G are graphs showing 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 during viral inactivation, Figure 4E shows the base flow during viral inactivation, Figure 4F shows high molecular weight species during viral inactivation, and Figure 4G shows the protein concentration after viral inactivation. [Figure 4-2] Continuation of Figure 4-1. [Figure 4-3] Continuation of Figure 4-2. [Figure 5-1] Figures 5A to 5D are graphs showing the post-capture operational performance of the methods and systems disclosed herein. Figure 5A shows residual protein A in the eluted product after capture, Figure 5B shows the protein concentration in the eluted product after capture, Figure 5C shows the ultraviolet chromatogram, and Figure 5D shows the residual host cell proteins in the eluted product after capture. [Figure 5-2] Continuation of Figure 5-1. [Figure 6-1]FIG. 6A is a schematic diagram of virus filtration of the methods and systems disclosed herein. FIGS. 6B-6G are graphs showing the virus filtration operation performance of the methods and systems disclosed herein. FIG. 6B shows the protein concentration after virus filtration, FIG. 6C shows the high molecular weight species after virus filtration, FIG. 6D shows the protein mass flux during virus filtration, FIG. 6E shows the filtrate volume flux during virus filtration, FIG. 6F shows the transmembrane pressure during virus filtration, and FIG. 6G shows the filter performance during virus filtration. [Figure 6-2] Continuation of FIG. 6-1. [Figure 6-3] Continuation of FIG. 6-2. [Figure 6-4] Continuation of FIG. 6-3. [Figure 7-1] FIGS. 7A-7E are graphs showing the ultrafiltration operation performance of the methods and systems disclosed herein. FIG. 7A shows the protein concentration and conversion after ultrafiltration, FIG. 7B shows the filter performance during ultrafiltration, FIG. 7C shows the inlet protein mass flux during ultrafiltration, FIG. 7D shows the permeate volume flux during ultrafiltration, and FIG. 7E shows the transmembrane pressure during ultrafiltration. [Figure 7-2] Continuation of FIG. 7-1. [Figure 8-1] FIGS. 8A-8E are graphs showing the diafiltration operation performance of the methods and systems disclosed herein. FIG. 8A shows the permeate volume flux during diafiltration, FIG. 8B shows the protein mass flux during diafiltration, FIG. 8C shows the transmembrane pressure during diafiltration, FIG. 8D shows the diafiltration ratio, and FIG. 8E shows the filter performance during diafiltration. [Figure 8-2] Continuation of FIG. 8-1. [Figure 8-3] Continuation of FIG. 8-2. [Figure 9-1]Figures 9A-9H are graphs showing the properties of a pharmaceutical substance 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 profile of the pharmaceutical substance, Figure 9D shows the osmotic pressure of the pharmaceutical substance, Figure 9E shows the pH of the pharmaceutical substance, Figure 9F shows the host cell proteins measured by ELISA of the pharmaceutical substance, Figure 9G shows the non-reduced purity of the pharmaceutical substance, and Figure 9H shows the residual Protein A of the pharmaceutical substance. [Figure 9-2] Continuation of Figure 9-1. [Figure 9-3] Continuation of Figure 9-2. [Figure 9-4] Continuation of Figure 9-3.

Mode for Carrying Out the Invention

[0011] The present disclosure relates to methods and systems for continuously producing recombinant proteins. In certain embodiments, the present disclosure relates to methods and systems for producing recombinant proteins using capture chromatography, post-capture chromatography, and ultrafiltration / diafiltration. The methods and systems described herein provide for the continuous and time-efficient production of recombinant proteins.

[0012] When used in accordance with the present disclosure, unless otherwise specified, all technical and scientific terms are to be understood as having the same meaning as commonly understood by one of ordinary skill in the art. Unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include singulars.

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

[0014] Furthermore, the present invention provides a manufacturing system for producing recombinant proteins, comprising: a first unit operation comprising a bioreactor containing host cells that produce recombinant proteins; 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 a first unit operation and a second unit operation, the fifth unit operation including a subsystem for pre-inactivating the virus. In some embodiments, the manufacturing system includes a sixth unit operation located between a third unit operation and a fourth unit operation, the sixth unit operation including a second subsystem for pre-filtration of the virus. In some embodiments, the manufacturing system includes a seventh unit operation including a third subsystem, the third subsystem including in-line excipients for formulating therapeutic drug substances.

[0016] In this specification, “substantially cell-free” means a sample that contains at least or about 90% of certain substances, such as mammalian cells (for example, at least or about 95%, 96%, 97%, 98%, or at least or about 99%, or about 100%).

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

[0018] In this specification, “liquid culture medium” refers to a liquid containing sufficient nutrients for 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 mammalian serum. In some embodiments, the liquid culture medium may not contain mammalian serum or other extracts (defined liquid culture medium). In some embodiments, the liquid culture medium may contain trace metals, mammalian growth hormones, and / or mammalian growth factors. Additional suitable liquid culture media are known and commercially available in the art.

[0019] In this specification, “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 immediately thereafter adding substantially the same 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 medium removed and added over a gradual period 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 exceeding 24 hours) (e.g., the rate of medium resupply on a daily basis). The percentage of medium removed and replaced each day may vary depending on the specific cells being cultured, the initial seeding density, and the cell density at a particular time.

[0020] In this specification, “fed-batch bioreactor” refers to a bioreactor containing multiple cells, to which nutrients necessary for cell growth and product formation are supplied intermittently or continuously by one or more feed streams.

[0021] In this specification, “clarified liquid culture medium” refers to a liquid culture medium obtained from bacterial or yeast cell cultures that is substantially free of bacterial or yeast cells (for example, at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% free).

[0022] In this specification, “recombinant protein” refers to immunoglobulins, protein fragments, engineered proteins, blood factors, nanobodies, or enzymes, and therefore also includes antibodies or antibody fragments.

[0023] In this specification, “unit operation” refers to a functional step that can be performed as a component of a system used in a method for producing recombinant proteins or a recombinant protein production process. For example, unit operations may include filtration (e.g., removal of contaminating bacteria, yeast viruses, or mycobacteria and / or certain substances from a liquid containing recombinant therapeutic protein), capture, removal of epitope tags, purification, retention or storage, polishing, virus inactivation, adjustment of the ionic concentration and / or pH of a liquid containing recombinant protein, and removal of unwanted salts.

[0024] In this specification, the terms “continuous method” or “continuous system” refer to a method or system in which liquid is continuously supplied to at least a portion of the unit operations. A unit operation is continuous if it can process a continuous flow input for an extended period of time. A continuous unit operation has a minimum internal retention volume. The output is continuous or discretized into small packets that are generated periodically. A method is continuous if it consists of integrated (physically connected) continuous unit operations with zero or minimal internal retention volume between them.

[0025] In this specification, “integrated method” refers to a method performed using structural elements that work together to achieve a specific result (e.g., the generation of recombinant proteins from liquid culture media).

[0026] In this specification, “elute” refers to the liquid eluten from a chromatography column or chromatography membrane containing a detectable amount of recombinant protein.

[0027] In this specification, "chromatographic medium" refers to the material packed into a chromatography column.

[0028] In some embodiments, the systems disclosed herein are enclosed systems. An enclosed system includes unit operations designed and operated to limit exposure to the external environment. Materials can be introduced into an enclosed system, but additions must be made in a manner that avoids exposure of the products to the indoor environment.

[0029] The methods and systems disclosed herein include capturing recombinant proteins using one or more capture chromatography systems and eluting the recombinant proteins from one or more capture chromatography systems to produce elutes containing the recombinant proteins. The chromatography media contained in one or more of the capture chromatography systems include capture mechanisms (e.g., protein A binding capture mechanisms, protein G binding capture mechanisms, antibody or antibody fragment binding capture mechanisms, substrate binding capture mechanisms, cofactor binding capture mechanisms). The resin may utilize a capture mechanism, a tag-binding capture mechanism, and / or an aptamer-binding capture mechanism. In some embodiments, one or more capture chromatography systems include a chromatography medium consisting of resin beads, a porous membrane, 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, one or more capture chromatography systems are periodic countercurrent chromatography systems (PCCS). In some embodiments, the PCCS includes two chromatography columns, or a modified AKTA system (GE Healthcare, Piscataway, NJ) capable of running up to, for example, four, five, six, seven, eight, or more columns. In some embodiments, the PCCS utilizes a column switching mechanism. The column switching 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 elapsed.

[0031] To capture recombinant proteins using a capture chromatography system, a series of chromatographic steps are required, including loading, washing, elution, and regeneration of the capture chromatography system.

[0032] In some embodiments, elutes from one or more capture chromatography systems are homogenized into a single mixture containing recombinant protein. In some embodiments, the homogenized single mixture is subjected to one or more post-capture chromatography systems to collect the output of a product containing recombinant protein. The post-capture chromatography systems are used to remove any remaining trace or small amounts of contaminants or impurities from the liquid containing recombinant protein close to the final desired purity. In some embodiments, one or more post-capture chromatography systems include a chromatography medium consisting 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, one or more post-capture chromatography columns are periodic countercurrent chromatography systems (PCCS).

[0033] To capture recombinant proteins using a post-capture chromatography system, a series of chromatography steps are required, including loading, washing, elution, and regeneration of the system.

[0034] In some embodiments, the method and system include ultrafiltration (UF) and / or diafiltration (DF) to further purify and concentrate the recombinant protein. UF / DF not only increases the concentration of the recombinant protein but can also replace the buffer salt with a specific formulation buffer. Ultrafiltration (UF) is a type of membrane filtration in which hydrostatic pressure presses a liquid against a semipermeable membrane. In some embodiments, UF is performed with tangential flow filtration (TFF), including 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 (permeate flow rate divided by inlet supply flow rate) in a single pass through the module is sufficiently large so that the system can operate without a retenate recycling loop. SPTFF is, for example, chromatography It can be used to reduce the process volume before the precipitation process, or to concentrate in-line between other unit operations. Zydney, Biotechnol. Bioeng. 113(3): pp. 465-475, (2016).

[0035] In this specification, “diafiltration” refers to a method of removing, replacing, or reducing the concentration of salts and buffering components from a solution containing proteins, peptides, nucleic acids, and other biomolecules, such as antibodies, using an ultrafiltration membrane. Continuous diafiltration (also known as constant-volume diafiltration) involves washing away salts (or other low molecular weight species) from the original buffer in the retained material by adding a new buffer, such as water or formulation buffer, to the retained material to form a formulation containing recombinant polypeptides. Generally, the new buffer is added at the same rate as the filtrate is produced so that the volume of the retained material 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 virus inactivation before being subjected to one or more post-capture chromatography systems. In some embodiments, virus inactivation includes inactivation with a solvent-surfactant solution, inactivation by heat, or inactivation by acidic pH.

[0037] Virus inactivation using solvents and surfactants involves subjecting a sample containing recombinant proteins to an organic solvent and a surfactant. Possible solvent-surfactant combinations include any combination of solvents and surfactants known to those skilled in the art, such as tri-n-butyl phosphate and TritonX-100™, Tween80™ and sodium cholate, and others.

[0038] Virus inactivation by thermal inactivation involves exposing a sample containing 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 approximately 49°C or higher, 50°C or higher, 51°C or higher, and above. In some embodiments, the sample is heated to temperatures between 45°C and 65°C.

[0039] The duration for heating the sample can be varied. For example, in some embodiments, the sample is heated to the target temperature for a period ranging from 1 minute to 6 hours. In some embodiments, the sample is heated to the target temperature for a period ranging from 10 to 180 minutes, 20 to 180 minutes, 20 to 60 minutes, or 20 to 40 minutes.

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

[0041] The methods and systems disclosed herein include inline monitoring, which includes detecting the level of recombinant protein detected by UV absorbance, detecting the flow rate, and / or detecting the volume of a liquid (e.g., buffer solution). Monitoring of recombinant protein concentration (e.g., monitoring performed by UV monitoring) can be determined by any tool capable of inline measurement of product concentration with feedback control.

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

[0043] In some embodiments, virus inactivation is carried out in a tubular flow reactor as described by Parker et al., Biotechnol. Bioeng. 115(3): pp. 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 viral filtration, is included after the collection of the product output from the post-capture chromatography system. The viral removal step is performed to remove small, non-enveloped viruses that are more resistant to viral inactivation treatment. In some embodiments, viral 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 pressurized loop is a hollow fiber virus filter. In some embodiments, the pressure vessel is a single-use, sealed, and sterilizable vessel. In this specification, the term “sterilizable” refers to a vessel formed from a material that conforms to known sterilization methods. In some embodiments, viral filtration is performed using dead-end filtration. In this specification, “dead-end filtration” refers to filtration in which the entire flow of the liquid being filtered passes through the filter without any recyclable or retained flow. In some embodiments, the virus removal filter is an ultrafiltration membrane or a nanofilter. An example of a suitable viral 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 the inlet of the second unit operation, the second unit operation includes an outlet connected to the inlet of the third unit operation, and the third unit operation includes an outlet connected to the inlet of the fourth unit operation. In some embodiments, the first unit operation includes an outlet connected to the inlet of the fifth unit operation, the fifth unit operation includes an outlet connected to the inlet of the second unit operation, the second unit operation includes an outlet connected to the inlet of the third unit operation, the third unit operation includes an outlet connected to the inlet of the sixth unit operation, and the sixth unit operation includes an outlet connected to the inlet of the fourth unit operation.

[0046] In some embodiments, the system disclosed herein includes a surge vessel between each of the first, second, third, and fourth unit operations. The surge vessel can hold any liquid culture before moving to the next unit operation.

[0047] The systems described herein may also include liquid conduits arranged between any unit operations. Suitable liquid conduits are polyethylene, polycarbonate, or plastic tubing. A liquid conduit may also include one or more of the following in any combination: one or more inline buffer preparation reservoirs, which are in liquid communication with the liquid conduit and arranged so that buffer stored in the inline buffer preparation reservoirs is added to the liquid present in the liquid conduit; and one or more filters, which are arranged within the liquid conduit so as to be able to filter the liquid present in the liquid conduit (e.g., to remove bacteria).

[0048] In some embodiments, the systems provided herein include a pumping system. The pumping 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.

[0049] In some embodiments, one or more excipients are generated after ultrafiltration / diafiltration. In addition to outputting material, it also generates therapeutic drug substances.

[0050] In this specification, “therapeutic drug substance” refers to a substance comprising recombinant proteins that have been thoroughly purified or isolated from contaminated proteins, lipids, nucleic acids (e.g., contaminated proteins, lipids, nucleic acids present in liquid culture media), host cells (e.g., derived from mammalian, yeast, or bacterial host cells), and biological contaminants (e.g., viral or bacterial contaminants), and which can be formulated into a pharmaceutical product without further substantial purification and / or decontamination steps.

[0051] In some embodiments, the systems disclosed herein are mounted on skids. Herein, “skid” refers to a three-dimensional solid structure that can function as a platform or support for the systems described herein. Mobility can be imparted to the system or a part thereof if the skid includes one or more structures that enable movement (e.g., wheels, rollers, etc.).

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

[0053] In some embodiments, the methods and systems described herein result in a net yield of recombinant protein in a therapeutic protein drug substance of at least about 5 g / day, 10 g / day, or 30 g / day, and at least 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, 10, 20, or 30 days, and at least about 280, 290, 300, 310, 320, 330, 340, 350, or 365 days.

[0054] The following embodiments illustrate specific embodiments of the Disclosure and their various uses. They 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 70 days using a 100L enhanced perfusion bioreactor conjugated with dual ATF. The culture medium containing the cell-free mAbs (collected) was continuously added to a 100L collection surge container for further processing.

[0056] Antibody-containing medium was captured on a pilot-scale steam-in-place PCC skid using two pre-packed and irradiated 1 L (8 × 20 cm) Protein A affinity chromatography columns in binding and elution MCC mode. The capture process was performed with one of the two columns always loaded from a collection surge container, ensuring that the average inlet flow rate of the capture process matched the average outlet flow rate of the perfusion bioreactor. Loading of the Protein A columns was controlled to approximately 3% breakthrough by Delta UV. Elutions from each capture column were collected in a 5 L mixed container before viral inactivation. The Protein A operation was integrated with a perfusion bioreactor on day 12 and continued for 58 consecutive days. Data summarizing the Protein A operation performance are shown in Figures 3A-3F.

[0057] Individual protein A elutes were mixed in a 5L mixing container to ensure a uniform flow for the virus inactivation unit operation. The effluent from the mixing container was used before the next protein A elution cycle. The entire container was processed automatically. Homogenized protein A eluate was adjusted in-line to the target pH of 3.6 by adding 1M acetic acid. The low-pH protein A eluate was then fed through a tubular flow reactor (TFR) with a defined minimum residence time of at least 30 minutes to completely inactivate the virus at low pH for a sufficient amount of time. The virus-inactivated protein A eluate was then adjusted in-line to the target pH of 4.5 by adding 0.75M sodium acetate. The adjusted virus-inactivated protein A eluate was then processed through a sterile-grade filter and continuously collected in a small surge container for further processing. The virus inactivation procedure was integrated with the preceding procedure on day 15 and performed continuously for 55 days. Data summarizing the performance of the virus inactivation procedure are shown in Figures 4A-4G.

[0058] The prepared virus-inactivated protein A eluate was further processed in two orthogonal chromatography operations using a mixed-mode anion exchange resin and a hydrophobic interaction chromatography resin, and operated in flow-through mode. In this processing example, there was no inter-process adjustment between the two post-capture chromatography operations, and the flow-through from the mixed-mode column could be directly loaded onto the hydrophobic interaction column. The post-capture apparatus consisted of two 0.2 L (5 × 10 cm) packed mixed-mode columns and two 0.2 L (5 × 10 cm) packed hydrophobic interaction columns, and was operated in MCC mode on the same PCC skid as the protein A operation. The flow of prepared virus-inactivated protein A eluate was continuously loaded onto one of the two parallel post-capture apparatuses, and the column loading was automated so that approximately the mass of two protein A eluates was loaded in each post-capture cycle.

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

[0060] The captured eluted material is operated in tangential flow mode at 0.1m 2The material was further processed with a Planova 20N (AK Bio) virus removal filter. The captured eluate was continuously transferred from the surge vessel to a pressurized loop including a pressure vessel, virus removal filter, sterile-grade filter, and a peristaltic pump driving recirculation, as shown in Figure 6A. The pressure in the loop was automated so that the net volumetric flow rate (permeate flux) passing through the virus filter was equal to the net volumetric flow rate discharged from the post-capture operation. The Planova 20N filter was replaced periodically based on preliminary virus validation studies. 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 performed continuously for 41 days. Data summarizing the operational performance of the virus filtration operation are shown in Figures 6B-6F.

[0061] Virus-filtered material, 0.065 m 2 The material was further processed by ultrafiltration using an ILC single-pass TFF (SP-TFF) cassette to concentrate it to the target mAb concentration of 65 g / L. The material was delivered from the surge container after viral filtration to the inlet of the SP-TFF cassette at a flow rate matched to the outlet flow rate of the viral filtration operation. The flow rate of the retained material from the SP-TFF cassette was automatically controlled using a feedback loop based on in-line measurement of the product concentration to maintain the target concentration. Next, the concentrated mAb flow was sent directly to the inlet of a single-pass diafiltration (SP-DF) cassette for in-line buffer exchange. The diafiltration buffer was delivered to the buffer inlet of the SP-DF cassette, and the buffer flow was automated to maintain a constant buffer-to-product flow ratio. The flow rate of the retained material was controlled to match the inlet flow rate. Next, the concentrated excipient solution was added in-line to formulate the retained material flow from the DF after buffer exchange, generating a pharmaceutical product. The pharmaceutical product was further processed with a sterile-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 performed continuously for 29 days. The formulation operation and drug substance generation were integrated on day 46 and performed continuously for 25 days. Six batches of drug substances were generated during the 25 days. Data summarizing the operational performance of ultrafiltration and diafiltration are shown in Figures 7A-7E and 8A-8D. Figures 9A-9H show the properties of the generated drug substances. [Examples]

[0062] Continuous production of recombinant proteins Recombinant monoclonal antibodies (mAbs) were produced over 27 days using a 100L enhanced perfusion bioreactor conjugated with dual ATF. The culture medium containing the cell-free mAbs (collected) was continuously added to a 100L collection surge container for further processing.

[0063] Antibody-containing medium was captured on a pilot-scale steam-in-place PCC skid using two pre-packed and irradiated 1 L (8 × 20 cm) Protein A affinity chromatography columns in binding and elution MCC mode. The capture process was performed so that one of the two columns was always loaded from the collection surge container, and the average inlet flow rate of the capture process matched the average outlet flow rate of the perfusion bioreactor. The loading of the Protein A columns was controlled to a breakthrough of approximately 3% by Delta UV. Elutions from each capture column were collected in a 5 L mixed container before viral inactivation. The Protein A operation was integrated with a perfusion bioreactor on day 12 and continued for 15 consecutive days.

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

[0065] The prepared virus-inactivated protein A eluate was further processed by two orthogonal chromatography operations using a mixed-mode anion exchange resin and a hydrophobic interaction chromatography resin, and operated in flow-through mode. The post-capture instrument 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, and was operated in MCC mode. The flow of the prepared virus-inactivated protein A eluate 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 independently up to their respective load capacities.

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

[0067] The captured eluted material is operated in tangential flow mode at 0.1m 2The samples were further processed with a Planova 20N (AK Bio) virus removal filter. The captured eluate was then passed through the outlet of the hydrophobic interaction column, through a pressure vessel, the virus removal filter, a sterile-grade filter, and recirculated. The material was continuously transferred to a pressurized loop containing a peristaltic pump that drove the ring. The pressure within the loop was automated so that the net volumetric flow rate (permeate flux) passing through the virus filter was equal to the net volumetric flow rate discharged from the post-capture operation. The virus-filtered material was continuously collected in a small surge container for further processing. The virus filtration operation was integrated with the preceding unit operation on day 14 and performed continuously for 13 days.

[0068] The virus-filtered material is then processed sequentially in a 2 x 0.1 m area. 2 The material was further processed by ultrafiltration using gamma-irradiated TFF capsules and operated in single-pass mode. The virus-filtered material was concentrated to the target mAb concentration of 110 g / L. The material was delivered from the surge container after virus filtration to the inlet of the TFF capsule at a flow rate matching the outlet flow rate of the virus filtration operation. The flow rate of the retained material from the TFF capsule was automatically controlled using a feedback loop based on in-line measurement of the product concentration to maintain the target concentration. Next, the concentrated mAb flow was directly delivered to the inlet of a gamma-irradiated single-pass diafiltration (SP-DF) cassette for in-line buffer exchange. The diafiltration buffer was delivered to the buffer inlet of the SP-DF cassette, and the buffer flow was automated to maintain a constant buffer ratio to the product flow. The flow rate of the retained material was controlled to match the inlet flow rate. Next, the concentrated excipient solution was added in-line to formulate the retained material flow of the DF with the exchanged buffer, generating a formulation active ingredient. The addition of the formulation buffer was controlled by a feedback loop and an in-line sensor that measured the level of excipients in the formulation flow. The active pharmaceutical ingredient was further processed through a sterile-grade filter to produce the drug substance, which was collected in single-use containers. Ultrafiltration and diafiltration operations were integrated with the preceding unit operations on day 18, and the formulation operation was integrated on day 20. Due to problems with the in-line sensor, the UF / DF and formulation operations did not reach a steady state before the end of the campaign.

[0069] While this disclosure has been described in terms of various embodiments, those skilled in the art will understand that variations and modifications may occur. Therefore, the appended claims are intended to cover all such equivalent variations that fall within the scope of the disclosure as described in the claims. Furthermore, the section headings used herein are for structural purposes only and should not be considered to limit the subject matter described.

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

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

Claims

1. A method for continuously producing recombinant proteins, (a) capturing recombinant proteins from a substantially cell-free sample using one or more capture chromatography systems, and eluting the recombinant proteins from one or more capture chromatography systems to produce an eluate containing the recombinant proteins, wherein the eluate is homogenized into a single mixture containing the recombinant proteins; (b) subjecting a homogenized single mixture to virus inactivation; (c) The homogenized single mixture from step (b) is subjected to one or more post-capture chromatography systems to collect the output of a product containing recombinant protein; (d) The output of the product of step (c) is subjected to virus filtration; (e) Purify the recombinant protein by subjecting the output of the second product of step (d) to ultrafiltration and diafiltration; The method comprising, wherein each step is performed as a unit operation, each unit operation is physically connected from step (a) to step (e), and the connected unit operations from step (a) to step (e) process a continuous flow containing recombinant protein from step (a) to step (e).

2. The method according to claim 1, wherein the virus inactivation includes inactivation by a solvent-surfactant solution, inactivation by heat, or inactivation by an acidic pH.

3. Inactivation by acidic pH is: (a) Adding one or more solutions to a homogenized single mixture to adjust the pH to a low level in line; (b) Incubating the prepared homogenized single mixture at a low pH while it becomes a virus-inactivating mixture. The method according to claim 2, including the method described in claim 2.

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

5. Substantially cell-free samples include perfusion bioreactors containing host cells that produce recombinant proteins, and fed-batch bioreactors containing host cells that produce recombinant proteins. The method according to claim 1, or removed from a clarified liquid culture containing host cells that produce recombinant proteins.

6. The method according to claim 1, wherein one or more capture chromatography systems include a chromatography medium comprising resin beads, a porous membrane, or nanofibers.

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

8. The method according to claim 7, wherein the affinity chromatography medium is a protein A-based resin.

9. The method according to claim 1, wherein one or more capture chromatography systems are periodic countercurrent chromatography systems (PCCS).

10. The method according to claim 3, wherein the incubation of a prepared, homogenized single mixture at a low pH for a certain period of time resulting in virus inactivation is carried out in a tubular flow reactor.

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

12. The method according to claim 1, wherein the virus filtration is performed within a pressurized loop.

13. The method according to claim 12, wherein the pressurized loop includes a pressure vessel, a virus removal filter, and a sterilization filter.

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

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

16. The method according to claim 1, wherein the virus filtration is performed using dead-end filtration.

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

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

19. The method according to claim 1 or 18, wherein diafiltration is performed using a single-pass diafiltration cassette.

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

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

Images