Bioreactor for antibody production

Abstract

The present invention relates to methods for making high titer antibody products. In particular, the present invention relates, in part, to improved serum-free animal cell culture media that can be used to produce proteins of interest. In addition, the invention further relates to a chromatography procedure for the successful isolation of an antibody product target of the present disclosure.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application incorporates by reference the following provisional patent applications and claims priority to and the benefits of the following provisional patent applications: Provisional Patent Application No. 63 / 315,897 filed on March 2, 2022; Provisional Patent Application No. 63 / 411,899 filed on September 30, 2022; Provisional Patent Application No. 63 / 417,873 filed on October 20, 2022; Provisional Patent Application No. 63 / 436,854 filed on January 3, 2023; and Provisional Patent Application No. 63 / 448,655 filed on February 27, 2023.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety. The .xml copy created on January 31, 2023 is named sequencelisting.xml and is 180,965 bytes in size. Technical Field

[0005] The present application relates to the production of recombinant proteins in highly productive cells using serum-free media for culture-scale production, as well as improved systems and methods for measuring and maintaining cell culture conditions, collecting and purifying proteins with reduced heterogeneity and improved quality and with higher yields. Background Art

[0006] The manufacturing process of therapeutic protein products should be optimized based on various factors, including (but not limited to) protein production conditions, protein structure and functional properties, and the desired final drug product. Even when the same therapeutic protein is used for the same final drug product, it may be necessary to change the protein production conditions to optimize the titer, batch, yield or quality of the product. In turn, increased and optimized titer, batch and yield require optimization of the collection and purification process to handle the increased titer and loading while maintaining acceptable quality attributes.

[0007] To support the growing demand for recombinantly produced protein pharmaceutical products, cell production methods have been developed that can produce higher titers and improved quality (including reduced heterogeneity and reduced sequence variants) while improving overall yield. Therefore, improved cell culture media are needed to meet these requirements and specialized cell conditions.

[0008] Additionally, measuring and maintaining optimized cell culture conditions is critical and requires specially designed equipment, including probes and sensors for measuring dissolved gases such as oxygen. However, the use of large-scale vessels and bioreactors can impact the equipment used to measure cell culture conditions, including its accuracy and maintenance requirements. Since the biopharmaceutical industry is strictly regulated, irregularities in measurements may require investigation, potentially requiring additional resources and delaying the manufacturing process. Therefore, there is a need for improved systems and methods for measuring dissolved gases in large-scale bioreactors that provide improved accuracy and reduce maintenance requirements.

[0009] After production, isolating highly concentrated proteins presents additional challenges, including limited processing volumes, filtration capabilities, chromatography column loading capacity, and potentially high drug substance viscosity, each of which can affect results such as material usage, time spent, product quality, batch purity, and batch yield. Therefore, new methods for collecting, purifying, and formulating high-titer therapeutic proteins have also been developed to address these challenges at each unit operation of the exemplary collection and purification process.

[0010] In order to be able to handle higher titer drug products, improved methods and systems for manufacturing, collecting and purifying high titer antibody drug products have been developed. For example, increased drug product titer and drug loading can exceed the capacity of certain chromatographic purification processes and change viscosity, which adversely affects the transfer of drug products along different chromatographic steps. Therefore, there is a need for improved methods that address not only individual purification steps but also the entire manufacturing process. Summary of the invention

[0011] The present disclosure provides, in part, an improved method for large-scale production of anti-IL4Rα antibodies in highly productive cells. Improved cell culture media and methods for maintaining optimal cell culture conditions have been developed to increase titer and improve quality. Improved methods have also been developed to collect and purify increased protein loads while increasing overall yield, reducing manufacturing and operational complexity, and improving the overall quality of the drug substance and formulated drug product.

[0012] In some embodiments, the anti-IL-4Rα antibody is Dupilumab. In one aspect, the anti-IL-4Rα antibody comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2. In one aspect, the anti-IL-4Rα antibody comprises three heavy chain complementary determining region (HCDR) sequences comprising SEQ ID NO: 3, 4 and 5 and three light chain complementary determining region (LCDR) sequences comprising SEQ ID NO: 6, 7 and 8.

[0013] In some embodiments, the protein of interest is an antigen binding protein, a blocking antibody, or a receptor antagonist (e.g., interleukin-4 receptor alpha (IL-4Rα)). In some aspects, the protein of interest is a protein having an Fc domain. Thus, in some aspects, the protein of interest is an antibody, such as a human antibody, a humanized antibody, a chimeric antibody, an antibody fragment (e.g., Fab or F(ab') 2 ), ScFv molecules, etc.

[0014] In some embodiments, the manufacturing process includes culturing cells capable of expressing an anti-IL4Rα antibody or receptor antagonist as described above in a cell culture medium comprising a chemically defined basal medium (e.g., a custom formulation or a commercially available basal medium). In another embodiment, the complete culture medium is serum-free ("serum-free" culture medium) and / or is free of hydrolysates. In another embodiment, the method includes the step of adding one or more point-of-use additives described below to the cell culture medium. In some aspects, the point-of-use additives described below may also be included in the cell culture medium at the beginning.

[0015] It has been found that polyamine-supplemented medium ("PS medium") increases cell viability and density, reduces cell doubling time, and allows cells grown in such supplemented medium to produce high titer proteins. It has been found that PS medium with or without serum and / or supplemented hydrolysate provides restored cell viability and density, cell doubling time, and high titer protein production.

[0016] In one embodiment, cells cultured in PS medium have an average doubling time of no more than 30 hours. In one aspect, the cell doubling time is no more than 24 hours. Likewise, including polyamines in serum-free medium allows cultured cells to reach a higher viable cell density (VCD) than when polyamines are not included.

[0017] In one embodiment, in the serum-free and / or hydrolysate-free cell culture medium, the culture medium comprises one or more polyamines selected from the group consisting of ornithine, putrescine, spermine, spermidine, or a combination thereof.

[0018] In one embodiment, the PS medium is serum-free and comprises ornithine at a concentration between 30 μM and 900 μM. In one aspect, ornithine is present in an amount of at least about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 540, 545, 550, 555, 560, 565, 568, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589 , 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 625, 630, 635, 640, 645, 650, 700, 750, 800, 850 or 900 μM (expressed in micromoles per liter) is present in the culture medium. Ornithine can be present at about 0.09 mM to about 0.9 mM. In some embodiments, the PS medium contains ≤ 7.5 g / L of hydrolysate. In some embodiments, the PS medium does not contain any hydrolysate.

[0019] In one embodiment, the PS medium is serum-free and comprises putrescine at a concentration between 30 μM and 900 μM. In one aspect, the putrescine is present in an amount of at least about 30, 40, 50, 60, 70, 80, 90, 100, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270 , 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 260, 365, 370, 375, 380, 385, 390, 395, 400, 405 or 410 μM concentration (expressed in micromoles / liter) is present in the culture medium. Putrescine can be present at about 0.20mM to about 0.714mM. In one embodiment, the PS culture medium contains 57mg / L±8.55mg / L of putrescine·2HCl and ≥15mg / L±2.25mg / L of ornithine·HCl. In some embodiments, the culture medium contains ≤7.5g / L of hydrolyzate. In some embodiments, the PS culture medium does not contain any hydrolyzate.

[0020] In one embodiment, the PS medium is serum-free and comprises spermine at a concentration between 10 μM and 900 μM. In one aspect, spermine is present at a concentration of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 540, 545, 550, 555, 560, 565, 568, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, In some embodiments, the PS medium comprises ≤ 7.5 g / L of hydrolyzate. In some embodiments, the PS medium does not contain any hydrolyzate.

[0021] In one embodiment, the PS medium is serum-free and comprises spermidine at a concentration between 10 μM and 900 μM. In one aspect, spermidine is present at a concentration of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 260, 365, 370, 375, 380, 385, 390, 395, 400, 405 or 410 μM concentration (expressed in micromoles / liter) is present in the culture medium. In one embodiment, the PS culture medium contains 57 mg / L ± 8.55 mg / L of spermine 4HCl and ≥ 15 mg / L ± 2.25 mg / L of spermidine HCl. In some embodiments, the culture medium contains ≤ 7.5 g / L of hydrolyzate. In some embodiments, the culture medium contains ≤ 16 g / L of hydrolyzate. In some embodiments, the PS culture medium does not contain any hydrolyzate.

[0022] In one embodiment, the culture medium comprises a mixture of amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof (notably, glutamine is an exception, which can be added back to the culture medium as a point-of-use addition).

[0023] In some embodiments, the culture medium comprises at least 40 ± 6 mM or at least 70 ± 10.5 mM amino acid or a mixture of amino acid salts. In one embodiment, the culture medium comprises at least 40 mM amino acid or a mixture of amino acid salts. In this or another aspect, the culture medium comprises at least 70 mM amino acid or a mixture of amino acid salts. In some embodiments, the whole process including the basal medium and the feed contains a total of at least 115 mM amino acid or a mixture of amino acid salts.

[0024] In some embodiments, the culture medium comprises one or more fatty acids. In one aspect, the culture medium comprises a mixture of fatty acids (or fatty acid derivatives) and alpha tocopherol. In one aspect, the fatty acids or fatty acid derivatives are selected from the group consisting of linoleic acid, linolenic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, lauric acid, behenic acid, capric acid, dodecanoic acid, caproic acid, lignoceric acid, myristic acid and caprylic acid, and may also include lipoic acid derived from the fatty acid.

[0025] In some embodiments, the culture medium comprises one or more nucleosides. In one aspect, the nucleoside is selected from the group consisting of adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof.

[0026] In some embodiments, the cell culture medium comprises one or more salts. In some aspects, the salt is selected from the group of divalent cations, such as calcium salts, magnesium salts, and combinations thereof. In one embodiment, the culture medium comprises calcium chloride, magnesium sulfate, and combinations thereof. In another aspect, the salt may also include a salt of a phosphate radical.

[0027] In some embodiments, the cell culture medium comprises any one or more of the following: NaHCO 3 , glutamine, insulin, glucose, CuSO 4 、ZnSO 4 、FeCl 3 、NiSO 4 、Na 4 EDTA, trisodium citrate (Na 3 In one aspect, the method comprises the step of adding to the cell culture medium one or more point-of-use chemicals selected from the group consisting of: NaHCO 3, glutamine, insulin, glucose, CuSO 4 、ZnSO 4 、FeCl 3 、NiSO 4 、Na 4 EDTA, trisodium citrate, and combinations thereof.

[0028] In some embodiments, the point-of-use supplement comprises taurine, phosphate esters, poloxamer 188, and combinations thereof. In some aspects, the point-of-use supplement can be included in the culture medium at the outset.

[0029] It has been found that including taurine in a cell culture medium increases the specific productivity of the cells and enables those cells to produce less ammonia byproducts. In one aspect, the cell culture medium is serum-free and comprises about 0.1 mM to about 10 mM taurine, about 1 mM to about 9 mM taurine, about 1 mM to about 8 mM taurine, about 1 mM to about 7 mM taurine, about 1 mM to about 6 mM taurine, about 1 mM to about 5 mM taurine, about 1 mM to about 4 mM taurine, about 1 mM to about 3 mM taurine, about 1 mM to about 2 mM taurine, about 0.1 mM to about 1 mM taurine, about 0.2 mM to about 1 mM taurine, about 0.3 mM to about 1 mM taurine, about 0.4 mM to about 1 mM taurine, or about 0.5 mM to about 1 mM taurine.

[0030] In some embodiments, the present disclosure provides a method for producing an anti-IL-4Rα antibody or an antigen-binding fragment thereof by taking the following steps: (1) introducing a nucleic acid sequence encoding a protein of interest into a cell; (2) selecting cells carrying the nucleic acid sequence; (3) culturing the selected cells in an embodiment of the serum-free cell culture medium described in the present disclosure; and (4) expressing the protein of interest in the cell, wherein the protein of interest is secreted into the culture medium. The biotherapeutic protein may be a blocking antibody or a receptor antagonist (e.g., blocking IL-4Rα and IL-13) and / or an antigen binding protein, which may comprise an Fc domain. In some embodiments, the antibody is a human, humanized, chimeric or non-human monoclonal antibody or antibody fragment.

[0031] In some embodiments, the cell is a mammalian cell, an avian cell, an insect cell, a yeast cell, or a bacterial cell. In one aspect, the cell is a mammalian cell suitable for producing a recombinant protein, such as a CHO cell or a derivative CHO-K1. In some aspects, the cell expresses a protein of interest, such as a biotherapeutic protein. In some aspects, the cell used to produce a protein is a mammalian cell capable of producing a biotherapeutic agent, such as a CHO, HEK293, and BHK cell, or any derivative thereof. In one embodiment, the cell is a CHO cell, such as a CHO-K1 cell.

[0032] In some embodiments, the cell culture medium: (1) is serum-free; (2) comprises a hydrolysate; (3) comprises one or more polyamines, including at least ornithine or spermine; (4) comprises at least about 40 mM or at least about 70 mM of a mixture of amino acids, including at least one of the following: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof; (5) comprises a mixture of tocopherols and fatty acids; (6) comprises a mixture of nucleosides, including at least one of the following: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; (7) comprises a salt of at least one of the following: calcium, magnesium, and phosphate; and (8) comprises at least one of the following point-of-use additives: NaHCO 3 , glutamine, insulin, glucose, CuSO 4 、ZnSO 4 、FeCl 3 、NiSO 4 、Na 4 EDTA, trisodium citrate, and combinations thereof.

[0033] In some embodiments, the cell culture medium: (1) is serum-free; (2) contains one or more polyamines, including at least ornithine or spermine; (3) contains at least about 40 mM or at least about 70 mM of a mixture of amino acids, including at least one of the following: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof; (4) contains a mixture of tocopherols and fatty acids; (5) contains a mixture of nucleosides, including at least one of the following: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; (6) contains salts, including at least one of the following: calcium salts, magnesium salts, phosphate salts, and combinations thereof; and (7) contains at least one of the following point-of-use additives: NaHCO 3, glutamine, insulin, glucose, CuSO 4 、ZnSO 4 、FeCl 3 、NiSO 4 、Na 4 EDTA, trisodium citrate, and combinations thereof.

[0034] In some embodiments, the cell culture medium: (1) is serum-free; (2) contains ≤ 7.5 g / L of hydrolysate; (3) contains 0.09 ± 0.014 mM, 0.3 ± 0.05 mM, 0.6 ± 0.09 mM, or 0.9 ± 0.14 mM ornithine; (4) optionally further contains 0.10 ± 0.03 mM, 0.20 ± 0.03 mM, 0.35 ± 0.06 mM, or 0.714 ± 0.11 mM putrescine, spermidine, or spermine; (5) contains at least about 40 mM or at least about 70 mM mixtures of amino acids including at least one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof; (6) mixtures comprising tocopherol and optionally fatty acids; (7) mixtures comprising nucleosides including adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; and (8) mixtures comprising calcium salts, magnesium salts, phosphates, and combinations thereof.

[0035] In some embodiments, the cell culture medium: (1) does not contain hydrolysis products; (2) contains ornithine at a concentration of at least 90 μM ± 14 μM; (3) and optionally contains putrescine, such as at least 150 μM ± 14 μM. In other embodiments, other polyamines, such as spermine, spermidine, etc. are contemplated to be within the scope of the present invention.

[0036] In some embodiments, the present disclosure provides a method for culturing cells in a serum-free medium, wherein the serum-free medium comprises: (1) 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM ornithine; (2) optionally 0.20±0.03 mM, 0.35±0.06, or 0.714±0.11 mM putrescine; (3) at least about 40 mM or at least about 70 mM of a mixture of amino acids, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, amine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; (4) tocopherol and a mixture of fatty acids; (5) a mixture of nucleosides including adenosine, guanosine, cytidine, uridine, thymidine and hypoxanthine; and (6) calcium salts, magnesium salts and phosphates, wherein the average doubling time of cells cultured according to this method does not exceed 24 hours or does not exceed one-third the doubling time of cells cultured using a similar cell culture medium containing less than 0.09±0.015 mM polyamine. In another embodiment, the cell culture is capable of achieving a viable cell density that is at least 3 times higher than a similar cell culture in a similar cell culture medium containing less than 0.09±0.014 mM ornithine (or less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine). In one embodiment, the culture medium comprises ≤ 7.5 g / L of hydrolysate; and in another embodiment, the culture medium does not contain hydrolysate.

[0037] In some embodiments, the protein of interest is produced by the following steps: (1) introducing a nucleic acid sequence encoding a protein of interest (such as an antibody or other antigen binding protein) into CHO cells; (2) selecting cells carrying the nucleic acid sequence; (3) culturing the selected cells in a serum-free cell culture medium, wherein the serum-free cell culture medium comprises: (a) 0.09±0.014, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM ornithine; (b) optionally 0.20±0.03 mM, 0.35±0.06, or 0.714±0.11 mM putrescine; (c) at least 40 mM or at least 70 mM of a mixture of amino acids. (d) a mixture of tocopherols and optionally fatty acids; (e) a mixture of nucleosides including one or more of adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; and (f) a salt including one or more of calcium salts, magnesium salts, phosphates, and combinations thereof; and (4) expressing a protein of interest in CHO cells, wherein the protein of interest is secreted into the culture medium. In some embodiments, the serum-free cell culture medium may include ≤7.5 g / L of hydrolysate; or in other embodiments, the hydrolysate is not included at all.

[0038] In some embodiments, the protein of interest is produced by the following steps: (1) introducing a nucleic acid sequence encoding a protein of interest (e.g., an antibody or other antigen binding protein) into CHO cells; (2) selecting cells carrying the nucleic acid sequence; (3) culturing the selected cells in a serum-free cell culture medium comprising: (a) 0.09±0.014, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM ornithine; (b) optionally 0.20±0.03 mM, 0.35±0.06, or 0.714±0.11 mM putrescine; (c) at least 40 mM or at least 70 mM of a mixture of amino acids. (d) a mixture of tocopherols and fatty acids; (e) a mixture of nucleosides including one or more of adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; and (f) a salt including one or more of calcium salts, magnesium salts, and phosphates; and (4) expressing a protein of interest in CHO cells, wherein the protein of interest is secreted into the culture medium. In some embodiments, the serum-free cell culture medium may include ≤7.5 g / L of hydrolysate; or in other embodiments, the hydrolysate is not included at all.

[0039] The present disclosure provides, in part, an improved seed train method for increasing titer. In one embodiment, the method is performed at 1.7×10 5 to 2.3×10 5 Compared with the standard initial viable cell density (VCD) in the range of 10 cells / mL, the initial VCD at each step (N-5 to N-1) increased to approximately 3.5×10 5 to about 5.43×10 5Cells / mL. In some aspects, the initial VCD (in N-5 to N-1) is about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 times higher than the alternative initial VCD in the standard seed expansion culture. In one aspect, the optimized seed expansion culture causes a 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 1%, 17%, 18%, 19%, or 20% increase in final titer (g / L). In one aspect, the peak lactate observed in the final production vessel is not substantially different from that of the standard seed expansion culture.

[0040] The present disclosure provides, in part, improved containers and bioreactors for measuring and controlling cell culture conditions, including dissolved gases, such as oxygen and carbon dioxide, which are important process parameters for maintaining healthy cell culture production processes within the biopharmaceutical industry.

[0041] The present disclosure provides, in part, improved methods and systems for air sparging, agitation, and measurement of dissolved gases. More specifically, the present disclosure provides optimized dissolved gas concentrations at each time interval, and improved bioreactors with optimized agitation rates and working volumes to provide the necessary agitation while minimizing shear stress.

[0042] In certain exemplary embodiments, starting volumes, air sparging, and agitation set points in vessels and bioreactors including various sensors along the seed-scale-up culture during the growth and production phases are optimized to improve process stability by minimizing shear stress on the cell culture and optimizing viable cell density (VCD), dissolved oxygen, carbon dioxide, and pH at each time.

[0043] In some embodiments, the sparging rate can be used to change the pCO within the cell culture. 2 The levels are used to modulate the acidic and basic variants of dupilumab in cultured cells expressing dupilumab.

[0044] The present disclosure also provides improved methods and systems for measuring dissolved gases. Electrochemical probes and optical probes can also be used to measure other dissolved gases, such as carbon dioxide. Compared to optical probes, electrochemical probes are more commonly used in containers and bioreactors because they have long been proven to have a wider linear range and faster response time than optical probes or sensors. However, electrochemical dissolved oxygen probes require a lot of maintenance in terms of frequent replacement of membranes and electrolyte solutions, and require long polarization times. In addition, it is known that electrochemical probes provide noisy readings due to bubble interference, and there is evidence of this in large-scale bioreactors provided in the examples below. The noise can cause offsets beyond the normal operating range, and due to strict supervision in the industry, the offset must be studied and confirmed by the quality assurance system of a biopharmaceutical company. Studies are conducted in the case of short-term high-side offsets that usually occur due to bubbling interference, thereby limiting the manufacturing source.

[0045] Although optical probes are less affected by bubble interference from stirring, agitation, and bubbling, and do not have the same electrolyte replacement and polarization requirements, optical probes are less accurate and less reactive than electrochemical probes. In addition, previous studies evaluating the use of optical probes have been limited to water monitoring applications and small-scale cell culture applications. The largest container used during testing of optical probes in the review literature was a 50L disposable bioreactor (Marvell et al., 2009), leaving a gap in the scientific literature reporting on the use of optical probes in large-scale stainless steel production (>1,000L) where performance issues of optical probes have not yet been resolved.

[0046] Therefore, the present disclosure provides an improved system and method for reducing signal noise in an electrochemical probe, which reduces or eliminates false alarms attributable to bubble interference. In addition, the present disclosure provides a system and method for improving the accuracy of an optical probe for measuring dissolved gases in large-scale containers and bioreactors, which requires less maintenance than conventional electrochemical probes and is not susceptible to bubble interference. As discussed in more detail below, containers and bioreactors with electrochemical and optical probes are developed with improved data processing methods to optimize the accuracy of measurements while minimizing maintenance by reducing or eliminating undesired excursion events beyond the normal operating range, which requires lengthy quality studies in the strictly regulated biopharmaceutical industry.

[0047] In one embodiment, the present disclosure provides a method of measuring VCD of cells cultured in a bioreactor by applying an electric field to cells cultured in the bioreactor; measuring capacitance; and correlating the measured capacitance with viable cell density.

[0048] In one embodiment, the protein of interest is capable of being produced at an average seven-day titer that is at least 7% higher, at least 14% higher, at least 80% higher, at least two-fold higher, or at least three-fold higher than the average seven-day titer produced by similar cells in a serum-free cell culture medium comprising less than 0.09±0.014 mM ornithine (or less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine).

[0049] In order to collect and purify the increased protein product, new and improved methods were developed that could not only accommodate the increased protein yield but also improve the overall yield percentage. The methods included, for example, optimizing collection pretreatment, protein A chromatography, viral inactivation, anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, virus retention filtration, concentration and diafiltration, and bulk drug conditioning, which are further described below.

[0050] The present disclosure provides a method for purifying anti-IL4Rα antibodies. In some exemplary embodiments, the method comprises: (a) subjecting the antibody to pretreatment; (b) collecting the antibody; (c) subjecting the antibody to affinity chromatography; (d) subjecting the antibody pooled from the eluate of step (c) to viral inactivation at a pH of about 3 to about 4.5 and then adjusting the pH to about 5 to about 8; (e) subjecting the antibody pooled from step (d) to anion exchange chromatography in flow-through mode; (f) subjecting the antibody pooled from the flow-through fraction of step (e) to cation exchange chromatography in binding and elution mode; (g) subjecting the antibody pooled from the eluate of step (f) to hydrophobic interaction chromatography in flow-through mode; and (h) subjecting the antibody pooled from the flow-through fraction of step (g) to virus retention filtration.

[0051] In one aspect, the antibody of step (h) is further subjected to concentration and diafiltration using a diafiltration buffer having a pH between 4.0 and 4.5.

[0052] In one aspect, the diafiltration buffer comprises about 4 mM acetate to about 6 mM acetate.

[0053] In one aspect, the collection pretreatment comprises subjecting the antibody to an instantaneous pH level of about 4.5 to 5.0 or about 4.0 to about 5.5, and then increasing the pH level to about 5.5 to 6.5.

[0054] In one aspect, the affinity chromatography is Protein A chromatography.

[0055] In one aspect, the collecting of step (b) comprises centrifugation and depth filtration.

[0056] In one aspect, the antibody is collected in a 3 kL to 25 kL bioreactor.

[0057] In one aspect, the loading pH of the Protein A column is between 7 and 8. In one aspect, the loading pH of the Protein A column is between 6 and 8. In one aspect, the loading pH of the Protein A column is about 6.

[0058] In an alternative exemplary embodiment, the method comprises: (a) culturing cells expressing an anti-IL-4Rα antibody or an antigen-binding fragment thereof; (b) subjecting the cells to an instantaneous pH level of about 4.0 to 5.5 or about 4.0 to about 5.5, followed by adjusting the pH to about 5.5 to 6.5; (c) collecting the cells by centrifugation to separate cell debris from a clarified medium containing the anti-IL-4Rα antibody or an antigen-binding fragment thereof; (d) subjecting the clarified medium to affinity chromatography; (e) subjecting the anti-IL-4Rα antibody or an antigen-binding fragment thereof pooled from the eluate in step (d) to viral inactivation at a pH of about 3 to about 4.5 and then adjusting the pH to about 5.5 to 6.5; to about 5 to about 8; (f) subjecting the anti-IL-4Rα antibodies or antigen-binding fragments thereof pooled from step (e) to anion exchange chromatography in flow-through mode; (g) subjecting the anti-IL-4Rα antibodies or antigen-binding fragments thereof pooled from the flow-through fraction of step (f) to cation exchange chromatography in bind and elute mode; (h) subjecting the anti-IL-4Rα antibodies or antigen-binding fragments thereof pooled from the eluate of step (g) to hydrophobic interaction chromatography in flow-through mode; and (i) subjecting the anti-IL-4Rα antibodies or antigen-binding fragments thereof pooled from the flow-through fraction of step (h) to virus retention filtration, thereby producing anti-IL-4Rα antibodies or antigen-binding fragments thereof.

[0059] In one aspect, the method further comprises subjecting the anti-IL-4Rα antibody or antigen-binding fragment thereof to ultrafiltration and diafiltration (UF / DF) after step (i).

[0060] In one aspect, the affinity chromatography is Protein A chromatography.

[0061] In one aspect, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises three heavy chain complementary determining region (HCDR) sequences comprising SEQ ID NOs: 3, 4 and 5 and three light chain complementary determining region (LCDR) sequences comprising SEQ ID NOs: 6, 7 and 8.

[0062] In one aspect, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2.

[0063] In one aspect, the anti-IL-4Rα antibody is dupilumab.

[0064] These and other aspects of the present disclosure will be better understood and appreciated when considered in conjunction with the following description and accompanying drawings. Although the following description indicates various embodiments and a number of specific details thereof, it is given by way of illustration only and is not limiting. Various substitutions, modifications, additions or rearrangements may be made within the scope of the present invention, and additional enumerated embodiments describing additional aspects of the present disclosure are provided below. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The structure of the antibody product dupilumab according to an exemplary embodiment is shown.

[0066] Figure 2 Graph showing the effect of insulin supplementation on the titer profile of dupilumab according to an exemplary embodiment.

[0067] Figure 3 Graph showing the effect of insulin supplementation on the viability of cells expressing dupilumab according to an exemplary embodiment.

[0068] Figure 4 Shown is the effect of insulin supplementation on the reduction of ammonia levels in a culture comprising cells expressing dupilumab according to an exemplary embodiment.

[0069] Figure 5 Shown are the effects of an insulin concentration of 15 mg / L and a feeding schedule on the growth rate, upper asymptote (maximum achievable titer), inflection point, and titer of a titer curve according to an exemplary embodiment.

[0070] Figure 6 The effect of insulin supplementation on high molecular weight species using a profiler for day 13 is shown according to an exemplary embodiment.

[0071] Figure 7 The effect of insulin supplementation on non-reducing purity levels using a profiler for day 13 is shown in accordance with an exemplary embodiment.

[0072] Figure 8 Shown is the effect of an insulin supplementation schedule on non-glycosylated heavy chain (NGHC) content for day 13 using a profiler according to an exemplary embodiment.

[0073] Fig. 9 Shown is the effect of tyrosine supplementation on titer and ammonia levels in cell cultures expressing dupilumab according to an exemplary embodiment.

[0074] Fig.10 Shown are the effects of phosphate redistribution strategies 6 and 8 on dupilumab titer (g / L) according to exemplary embodiments.

[0075] Fig.11Phosphate redistribution profiles are shown for days 0, 2, 4, 6, and 8 according to an exemplary embodiment.

[0076] Fig. 12A A contour plot showing a Central Composition Design (CCD) model for predicting host cell protein clearance factors (CF) according to an exemplary embodiment.

[0077] Fig. 12B A contour plot showing a CCD model predicting normalized supernatant turbidity (NTU) according to an exemplary embodiment.

[0078] Fig.13A Contour plot showing a CCD model for predicting host cell protein clearance factors according to an exemplary embodiment.

[0079] Fig. 13B A contour plot showing a CCD model predicting normalized supernatant turbidity (NTU) according to an exemplary embodiment.

[0080] Fig.14 Shown are initial and final viable cell densities (VCD) of an average standard seed-expanded culture and an optimized seed-expanded culture with an increase in initial VCD compared to a standard seed-expanded culture according to an exemplary embodiment.

[0081] Fig.15 Shown is the reduction in biomass in a 10 kL production bioreactor for cells cultured using an optimized seed-expansion culture with increased initial VCD compared to a standard seed-expansion culture, according to an exemplary embodiment.

[0082] Fig.16A and Fig. 16B Shown are final titers (g / L) of an optimized seed expansion culture (Sample 1) with increased initial VCD compared to standard seed expansion cultures (Samples 2 to 9) according to an exemplary embodiment.

[0083] Fig.17 Shown is a bivariate linear regression of on-line capacitance measurements and off-line VCD measurements during seed expansion culture of cell line A (CLA) according to an exemplary embodiment of the invention.

[0084] Fig.18 A bivariate linear regression of CLA during the N-3, N-2, and N-1 seed expansion culture stages and on-line capacitance measurements and off-line VCD measurements is shown according to an exemplary embodiment of the present invention.

[0085] Fig.19 The predicted values ​​of CLA during the N-3, N-2 and N-1 seed expansion culture stages and along-line VCD measurements are shown according to an exemplary embodiment of the present invention, using Fig.21 The VCD values ​​were determined using the online capacitance measurements and the correlation equation presented in Figure 2. The points (circles) indicate the VCD values ​​determined offline using the bioanalyzer.

[0086] Fig. 20 A bivariate linear regression of on-line capacitance measurements and off-line VCD measurements during seed expansion culture of cell line B (CLB) according to an exemplary embodiment of the invention is shown.

[0087] Fig.21 Bivariate linear regression of online capacitance measurements and offline VCD measurements of CLBs during the N-3, N-2, and N-1 seed expansion culture stages according to an exemplary embodiment of the present invention is shown.

[0088] Fig. 22 Predicted values ​​of CLB during the N-3, N-2 and N-1 seed expansion culture stages and along-line VCD measurements according to an exemplary embodiment of the present invention are shown, which are determined using along-line capacitance measurements and correlation equations presented in Figure 24. Points (circles) indicate VCD values ​​determined offline using a bioanalyzer.

[0089] Fig.23 Bivariate linear regression of CLA and CLB during seed expansion culture and with on-line capacitance measurements and off-line VCD measurements according to an exemplary embodiment of the invention is shown.

[0090] Fig.24A , Fig. 24B and Fig.24C The predicted values ​​of CLA during the N-3, N-2 and N-1 seed expansion culture stages and along-line VCD measurements are shown according to an exemplary embodiment of the present invention, using Fig.17 The in-line capacitance measurements presented in Fig.17 ( Fig.24A ), Fig. 20 ( Fig. 24B )and Fig.23 ( Fig.24C ) were determined using the correlation equation presented in ). Points (circles) indicate VCD values ​​determined offline using a bioanalyzer.

[0091] Fig.25A , Fig.25B and Fig.25C The predicted values ​​(black trace) of CLB during the N-3, N-2 and N-1 seed expansion culture stages and along-line VCD measurements according to an exemplary embodiment of the present invention are shown using Fig.21 The in-line capacitance measurements presented in Fig. 20 ( Fig.25A ), Fig.17 ( Fig.25B )and Fig.23 ( Fig.25C ) were determined using the correlation equation presented in ). Points (circles) indicate VCD values ​​determined offline using a bioanalyzer.

[0092] Fig.26A and Fig.26B The predicted values ​​of the online VCD measurements of CLB during the seed expansion and production phases according to an exemplary embodiment of the present invention are shown using Fig. 20 The linear capacitance measurements and correlation equations presented in Figure 1 are used to determine the linear capacitance. Figures A and B show data from two independent cultures, each of which used a single probe to perform the linear capacitance measurements. Fig.26A In , the line depicts the predicted value of the VCD measurement along the line, and the points (circles) indicate the VCD values ​​determined offline using the bioanalyzer. Fig.26B In the figure, the line depicts the predicted values ​​of the VCD measurements along the line, and the dots (circles) indicate the VCD values ​​determined offline using the bioanalyzer.

[0093] Fig.27A Shown are host cell protein values ​​and Dupilumab yield after acid precipitation of Dupilumab cell culture fluid using 1 M phosphoric acid according to an exemplary embodiment.

[0094] Fig.27B Shown are host cell protein values ​​and dupilumab yields after acid precipitation of dupilumab cell culture fluid using 1.75 M acetic acid according to an exemplary embodiment.

[0095] Fig.28 Schematic diagram showing an exemplary overview regarding the use of depth filters, fine filters and protection filters according to an exemplary embodiment.

[0096] Fig.29 A predictive profiler that models factors and reactions studied in a centrifuge characterization study is shown according to an exemplary embodiment.

[0097] Fig.30 A schematic diagram showing exemplary purification steps of an optimized process according to an exemplary embodiment.

[0098] Fig.31 A schematic diagram showing exemplary purification steps of an optimized process according to an exemplary embodiment.

[0099] Fig.32 Observations from a model generated for a multivariate study of affinity capture and viral inactivation risk factors and responses are shown according to an exemplary embodiment.

[0100] Fig.33Shown is the HCP content in the affinity capture pool when various affinity wash buffers are used according to exemplary embodiments.

[0101] Fig.34 Shown are HCP contents after AEX or HIC treatment downstream of affinity capture chromatography using various affinity wash buffers according to exemplary embodiments.

[0102] Fig.35 Shown are protein yields and HMW species in affinity capture pools when various affinity wash buffers are used according to exemplary embodiments.

[0103] Fig.36 Observations from a model generated from a multivariate study of AEX risk factors and responses are shown in accordance with an exemplary embodiment.

[0104] Fig.37 Observations from a model generated from a multivariate study of CEX risk factors and responses are shown in accordance with an exemplary embodiment.

[0105] Fig.38 Observations from a model generated from a multivariate study of HIC risk factors and responses are shown according to an exemplary embodiment.

[0106] Fig.39 Observations from a model generated from a multivariate study of VRF risk factors and responses are shown in accordance with an exemplary embodiment.

[0107] Fig.40 Observations from a model generated from a multivariate study of UF / DF risk factors and responses are shown in accordance with an exemplary embodiment.

[0108] Fig.41 Shown are bioreactor titers of a dupilumab 500L confirmation batch compared to a 10,000L process performance verification batch and a 2L pilot scale production according to an exemplary embodiment.

[0109] Fig.42 The step yields of unit operations are shown for a 500L confirmation batch compared to a 10,000L process performance verification batch according to an exemplary embodiment.

[0110] Fig.43 Shown is the HMW content of a unit operation for a 500L confirmation batch compared to a 10,000L process performance verification batch according to an exemplary embodiment.

[0111] Fig.44 The HCP content of the unit operation for a 500L confirmation batch compared to a 10,000L process performance verification batch according to an exemplary embodiment is shown.

[0112] Fig.45 An example of step-wise agitation and air sparging rate over time is shown according to an exemplary embodiment.

[0113] Fig.46 An example of applying an Agile filter to filter a cell culture signal at a 59 second sampling rate and about a 10 minute sample window according to an exemplary embodiment is shown. Mark "A" shows that the low side offset is retained for the smoothed signal. Mark "B" shows good agreement between the smoothed signal and the unfiltered signal for expected process disturbances.

[0114] Fig.47 An example of applying an Agile filter to filter a cell culture signal at a 59 second sampling rate and about a 33 minute sample window according to an exemplary embodiment is shown. Label "A" shows that the low side offset is removed for the smoothed signal. Label "B" shows good agreement between the smoothed signal and the unfiltered signal for expected process disturbances.

[0115] Fig.48 An example of signal filtering of a cell culture using a Savitzky-Golay filter at a 59 second sampling rate and approximately a 33 minute sample window is shown in accordance with an exemplary embodiment. Label "A" shows that noise remains for the filtered signal. Label "B" shows good agreement between the smoothed and unfiltered signals for expected process disturbances.

[0116] Fig.49 According to an exemplary embodiment, Figure 46 to Figure 48 Superposition comparison of the filtered signals.

[0117] Fig.50 Shown is the DO run 1 trend according to an exemplary embodiment (light line is electrochemical, medium line is optical probe B, darkest line is optical probe A, dashed lines: set point and upper and lower normal operating range limits).

[0118] Fig.51 Shown is the run 2 dissolved oxygen trend according to an exemplary embodiment (light line is electrochemical, medium line is optical probe B, darkest line is optical probe A, dashed lines: set point and upper and lower normal operating range limits).

[0119] Fig.52 Shown is the dissolved oxygen trend for Run 3 according to an exemplary embodiment (light line is electrochemical, medium line is optical probe B, darkest line is optical probe A, dashed lines: set point and upper and lower normal operating range limits).

[0120] Fig.53Shown is the dissolved oxygen run 4 trend according to an exemplary embodiment (light line is electrochemical, medium line is optical probe B, darkest line is optical probe A, dashed lines: set point and upper and lower normal operating range limits).

[0121] Fig.54 Dissolved oxygen data generated using optical probe B with an anti-bubble optical cap for runs 1 to 3 and a standard optical cap for run 4 is shown in accordance with an exemplary embodiment.

[0122] Fig.55 Shown is dissolved oxygen data as measured at the 'start', 'middle' and 'end' time periods of run 2 as detected as part of the excursion assessment in accordance with an exemplary embodiment (where the light line is electrochemistry, the medium line is optical probe B, the darkest line is optical probe A, and the bottom line is electrochemistry with an Agile filter (59s sampling rate and preset sample window)).

[0123] Fig.56 Shown is the 1st round of Probe B offset correction according to an exemplary embodiment (wherein the medium color line is optical Probe B, the darkest line is optical Probe B adjusted up by 3.0%, the light color line is electrochemistry with Agile filter (59s sampling rate and preset sample window), dashed lines: set point and upper / lower normal operating range limits).

[0124] Fig.57 Shown is the 3rd round of Probe B offset correction according to an exemplary embodiment (wherein the medium color line is optical Probe B, the darkest line is optical Probe B adjusted up by 3.0%, the light color line is electrochemistry with Agile filter (59s sampling rate and preset sample window), dashed lines: set point and upper / lower normal operating range limits).

[0125] Fig.58A and Fig.58B An example of varying the dextrose concentration at different times is shown.

[0126] Fig.59 Figure 5 shows the pCO of culture media with different sparging conditions (in mmHg) as a function of treatment time (days) according to an exemplary embodiment. 2 Profile (y-axis). Medium pCO2 was chosen as midpoint control.

[0127] Fig.60 Observations from a model generated for a multivariate study of risk factors and responses for mixed mode chromatography (MMC) using Capto Adhere resin according to an exemplary embodiment are shown.

[0128] Fig.61Observations from a model generated for a multivariate study of MMC risk factors and response using PPA HyperCel resin, according to an exemplary embodiment, are shown. DETAILED DESCRIPTION

[0129] The manufacturing process of therapeutic protein products (e.g., monoclonal antibody drugs) should be optimized based on various factors, including (but not limited to) recombinant host cell characteristics, protein production conditions, protein structure and functional properties, and the desired final drug product. In order to support the growing demand for recombinantly produced protein drug products, cell lines and cell culture media capable of producing higher titers and larger batches with improved quality and yield have been developed.

[0130] Even when using the same therapeutic protein for the same final drug product, it may be necessary to change protein production conditions to optimize the titer, batch, yield or quality of the product. Applicants have discovered that the addition of polyamines (such as ornithine, putrescine, spermine and spermidine, or one or a combination thereof) to the culture medium ("PS medium") improves viable cell density, cell doubling time and dupilumab protein production by cells in cell culture relative to serum-free medium containing little or no polyamines (such as ornithine, putrescine, spermine and spermidine) ("non-PS medium").

[0131] Conventional methods for collecting and purifying protein drug products rely on a series of filtration and chromatographic steps, which can be limited in the ability to use similar-sized equipment to handle increased titers and corresponding batches, protein concentrations, and drug product viscosity while maintaining purity and quality standards. For example, when moving a drug product with a higher protein concentration, a pump previously used to move the drug product may not pass due to increased viscosity levels. Techniques have been developed to reduce sample viscosity, such as adding arginine, a hydrophobic salt. However, adding arginine during collection and purification, especially during concentration and diafiltration, can result in the loss of an unknown amount of arginine in subsequent processing steps, which must then be measured and compensated for use in more downstream steps, and may increase the variation of the final drug product. Therefore, there is a need for systems and methods for collecting high-titer antibody drug products while maintaining product consistency, high yield, and quality.

[0132] The disclosure herein provides improvements to manufacturing, collection and purification methods to improve the production of high titer antibody products. In some exemplary embodiments, the high titer antibody product of interest is dupilumab.

[0133] Dupilumab is a human monoclonal immunoglobulin G4 (IgG4) antibody expressed and produced by Chinese hamster ovary cells (CHO-K1) that target interleukin-4 receptor subunit α (IL-4R-α). After cell culture, collection, isolation and purification, it is prepared for administration together with, for example, histidine, histidine hydrochloride, arginine hydrochloride, polysorbate 80, sodium acetate, glacial acetic acid, sucrose and water.

[0134] Dupilumab inhibits interleukin-4 (IL-4) and interleukin-13 (IL-13) signaling by specifically binding to the IL-4 receptor alpha (IL-4Rα) subunit shared by the IL-4 and IL-13 receptor complexes. Dupilumab inhibits IL-4 signaling via the type I receptor and IL-4 and IL-13 signaling via the type II receptor. Blocking IL4Rα with dupilumab inhibits IL-4 and IL-13 cytokine-induced inflammatory responses, including the release of proinflammatory cytokines, chemokines, nitric oxide, and IgE. By blocking the obligate shared components of the IL-4 / IL-13 receptor complex, dupilumab inhibits IL-4 and IL-13 signaling, key disease drivers of type II inflammation, and provides clinical benefit and long-term disease control without the side effects typically observed with existing non-selective systemic immunosuppressants.

[0135] Dupilumab received its first global marketing authorization in the United States on March 28, 2017, for the treatment of moderate to severe atopic dermatitis (AD) in adults, followed by marketing authorization in the European Union on September 28, 2017, in Japan on January 19, 2018, and subsequently in multiple other countries. Since then, Dupilumab has been approved for the treatment of other diseases associated with type II inflammation, including moderate to severe asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), eosinophilic esophagitis (EoE), chronic spontaneous urticaria (CSU), and prurigo nodularis (PN). In addition, subcutaneous administration of dupilumab is currently being developed for the treatment of patients with type 2 inflammatory diseases, including AD (pediatric patients (6 months to <6 years)), asthma (pediatric patients (6 months to <6 years)), chronic obstructive pulmonary disease (COPD), allergic fungal sinusitis (AFRS), chronic sinusitis without nasal polyps (CRSsNP), allergies (e.g., grass allergy, peanut allergy, and dairy allergy), bullous pemphigoid, hand and foot atopic dermatitis, cold urticaria, chronic induced urticaria, ulcerative colitis, chronic pruritus of unknown origin, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, bronchiectasis, alopecia areata, and allergic rhinitis.

[0136] In order to meet the high demand for dupilumab, a new manufacturing process was developed, as described further below. For example, the new manufacturing process allows for the production and purification of 150 mg / mL and 175 mg / mL of dupilumab formulated drug substance (FDS) at 10,000L scale and 25,000L scale.

[0137] Description of terms

[0138] Unless otherwise described, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Methods and materials similar or equivalent to those described herein can be used to practice the specific embodiments described herein. All disclosures mentioned are hereby incorporated by reference in their entirety.

[0139] The term "a" should be understood to mean "at least one", and the terms "about" and "approximately" should be understood to allow for standard deviations as would be understood by one of ordinary skill in the art, and when ranges are provided, the endpoints are included. As used herein, the terms "include", "includes", and "including" are intended to be non-limiting and should be understood to mean "comprise", "comprises", and "comprising", respectively.

[0140] As used herein, in the context of protein preparation, the term "upstream process technology" refers to activities involved in the production of proteins from cells in cell culture. As used herein, the term "cell culture" refers to methods for generating and maintaining a population of host cells capable of producing a recombinant protein of interest, as well as methods and techniques for optimizing the production and collection of a protein of interest. For example, once an expression vector has been incorporated into an appropriate host cell, the host cell can be maintained under conditions suitable for expression of the relevant nucleotide coding sequence and collection and production of the desired recombinant protein.

[0141] The present disclosure provides a serum-free medium, which is suitable for culturing cells and producing biopharmaceutical APIs. "Serum-free" is applicable to cell culture media that do not contain animal serum (such as fetal bovine serum). The serum-free medium may contain ≤7.5g / L of hydrolyzate, such as soybean hydrolyzate. The present disclosure also provides a chemically defined medium (CDM), which is not only serum-free but also free of hydrolyzate.

[0142] "Hydrolysate-free" applies to cell culture media that do not contain exogenous protein hydrolysates, such as animal or plant protein hydrolysates, including, for example, peptone, tryptone, and the like.

[0143] "Cell culture" or "culture" means the growth and reproduction of cells outside a multicellular organism or tissue. Culture conditions suitable for mammalian cells are known in the art. See, for example, Animal Cell Culture: A Practical Approach, D. Rickwood, Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid matrix at the same time. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, flasks or stirred tank bioreactors with or without microcarriers and operated in batch, batch feeding, continuous, semi-continuous or perfusion mode can be used for mammalian cell culture. Cell culture medium or concentrated feed medium can be added to the culture continuously or at certain time intervals during the culture period. For example, the culture can be fed once a day, every other day, every three days, or can be fed when the concentration of the monitored specific culture medium component falls outside the desired range.

[0144] As used herein, the term "chemically defined medium" or "chemically defined media" (both abbreviated as "CDM") refers to a synthetic growth medium in which the identities and concentrations of all components are defined. Chemically defined media do not contain bacteria, yeast, animal or plant extracts, animal serum or plasma, but may contain components derived from individual plants or animals (e.g., proteins, polypeptides, etc.). Chemically defined media may contain inorganic salts required to support growth, such as phosphates, sulfates, etc. The carbon source is defined and is typically a sugar, such as glucose, lactose, galactose, etc., or other compounds, such as glycerol, lactate, acetate, etc. Although some chemically defined media also use phosphate as a buffer, other buffers may also be used, such as sodium bicarbonate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), citrate, triethanolamine, etc. Examples of commercially available chemically defined media include, but are not limited to, various Dulbecco's Modified Eagle's (DME) media (Sigma-Aldrich Co; SAFC Biosciences, Inc.), Ham's Nutrient Mixture (Sigma-Aldrich Co; SAFC Biosciences), various EX-CELL media (Sigma-Aldrich Co; SAFC Biosciences), various IS CHO-CD media (FUJIFILM Irvine Scientific), combinations thereof, and the like. Methods for preparing chemically defined media are known in the art, for example, U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007 / 077217, and U.S. Patent Application Publication Nos. 2008 / 0009040 and 2007 / 0212770, the entire teachings of which are incorporated herein by reference.

[0145] As used herein, the term "recombinant host cell" (or simply "host cell") includes cells into which a recombinant expression vector encoding a protein of interest is introduced. It should be understood that such terms are intended not only to refer to specific individual cells, but also to the offspring of such cells. Since certain modifications may occur in offspring due to mutations or environmental influences, such offspring may not actually be consistent with the parental cell, but are still included in the scope of the term "host cell" as used herein. In one embodiment, the host cell includes prokaryotes and eukaryotic cells selected from any kingdom of life. In one aspect, eukaryotic cells include protists, fungi, plants, and animal cells. In another aspect, the host cell includes eukaryotic cells, such as plant and / or animal cells. The cell may be a mammalian cell, a fish cell, an insect cell, an amphibian cell, or an avian cell. In a specific aspect, the host cell is a mammalian cell. A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and other repositories and commercial suppliers. Cells that can be used in the methods of the present invention include, but are not limited to, MK2.7 cells, PER-C6 cells, Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCCCL-61) (Chasin et al., 1986, Somatic Cell and Molecular Genetics (Som. Cell Molec. Genet.), 12: 555-556; Kolkekar et al., 1997, Biochemistry (Biochemistry), 36: 10901-10909; and WO 01 / 92337A2), dihydrofolate reductase negative CHO cells (CHO / -DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77: 4216) and dp12.CHO cells (U.S. Patent No. 5,721,121); monkey kidney cells (CV1, ATCC CCL-70); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); HEK293 cells, Sp2 / 0 cells, 5L8 fusion tumor cells, Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkat cells ( cells), THP-1 cells, Sp2 / 0 cells, primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), and established cell lines and strains thereof (e.g., human embryonic kidney cells (e.g., HEK293 cells, or HEK293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); mouse Seth Rho cells (TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCCCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCCCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NY Acad. Sci., 383:44-68); MCR 5 cells; FS4 cells; PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS180 cells, LS174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK. 2 cells, clones M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK 1 Cells, PK(15) cells, GH 1 Cells, GH 3cells, L2 cells, LLC-RC 256 cells, MH 1 C 1 cells, XC cells, MDOK cells, VSW cells and TH-I, B1 cells or their derivatives), fibroblasts from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoids, tonsil, bone marrow and blood), spleen), and fibroblasts and fibroblast-like cell lines (e.g., TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl 1 cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C 3 H / IOTI / 2 cells, HSDM 1 C 3 cells, KLN205 cells, McCoy cells, mouse L cells, line 2071 (mouse L) cells, LM line (mouse L) cells, L-MTK (mouse L) cells, NCTC strains 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntac cells, SIRC cells, C II Cells and Jensen cells, or derivatives thereof), or any other cell type known to those skilled in the art. In some aspects, the cells used in the present invention are CHO cells, HEK293 cells, BHK cells, or derivatives thereof.

[0146] As used herein, the term "host cell protein" (HCP) includes proteins derived from host cells and may not be related to the desired protein of interest. Host cell proteins may be process-related impurities, which may be derived from the manufacturing process and may include three main categories: derived from the cell matrix, derived from the cell culture, and derived from the downstream. Impurities derived from the cell matrix include, but are not limited to, proteins derived from the host organism and nucleic acids (host cell genome, vector or total DNA). Impurities derived from cell culture include, but are not limited to, inducers, antibiotics, serum, and other culture medium components. Impurities derived from downstream include, but are not limited to, enzymes, chemical and biochemical treatment reagents (e.g., cyanogen bromide, guanidine, oxidants and reductants), inorganic salts (e.g., heavy metals, arsenic, non-metallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachates.

[0147] As used herein, the term "liquid chromatography" refers to a process in which a biological / chemical mixture carried by a liquid can be separated into components as it circulates (or flows into) a stationary liquid or solid phase due to the differential distribution of the components. Non-limiting examples of liquid chromatography include reversed phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed mode chromatography. In some aspects, a sample containing at least one protein or peptide digest of interest may undergo any one or a combination of the aforementioned chromatographic methods. Analytes separated using chromatography will be characterized by a unique retention time, reflecting the speed at which the analyte moves through the chromatographic column. Chromatograms can be used to compare analytes, which plot the retention time on one axis and the measured signal on the other axis, wherein the measured signal can be generated by, for example, UV detection or fluorescence detection.

[0148] As used herein, the term "mass spectrometer" includes a device capable of identifying specific molecular species and measuring their accurate mass. The term is intended to include any molecular detector by which a polypeptide or peptide can be characterized. A mass spectrometer may include three main parts: an ion source, a mass analyzer, and a detector. The function of the ion source is to produce gas phase ions. Analyte atoms, molecules, or clusters may be transferred into the gas phase and ionized simultaneously (as in electrospray ionization) or via separate processes. The choice of ion source depends on the application.

[0149] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. As used herein, the term "tandem mass spectrometry" includes techniques in which structural information about sample molecules is obtained by using multi-stage mass selection and mass separation. The prerequisite is that the sample molecules are converted into the gas phase and ionized so that fragments are formed in a predictable and controllable manner after the first mass selection step. MS / MS or MS 2 This can be accomplished by first selecting and isolating precursor ions (MS 1) and fragment it to obtain meaningful information. Tandem MS has been successfully performed with a wide variety of analyzer combinations. Which analyzers are combined for a certain application can be determined by several different factors, such as sensitivity, selectivity and speed, as well as size, cost and availability. The two main categories of tandem MS methods are spatial tandem and time tandem, but there are also hybrids, in which the time tandem analyzer is in space or coupled to the spatial tandem analyzer. The spatial tandem mass spectrometer includes an ion source, a precursor ion activation device and at least two non-capture mass analyzers. The specific m / z separation function can be designed so that ions are selected in one part of the instrument, dissociated in the middle zone, and the product ions are subsequently transmitted to another analyzer for m / z separation and data acquisition. In the time tandem, the mass spectrometer ions produced in the ion source can be trapped, separated, fragmented and m / z separated in the same physical device.

[0150] Peptides identified by mass spectrometry can be used as surrogate representatives of intact proteins and their post-translational or other modifications. They can be used for protein characterization by correlating experimental and theoretical MS / MS data, the latter data being generated from possible peptides in a protein sequence database. Characterization includes, but is not limited to, sequencing amino acids of protein fragments, determining protein sequencing, determining protein de novo sequencing, locating post-translational modifications, or identifying post-translational modifications, or comparability analysis, or a combination thereof.

[0151] In some exemplary aspects, a mass spectrometer can be used for nano-electrospray or nano-spray. As used herein, the term "nano-electrospray" or "nano-spray" refers to electrospray ionization at very low solvent flow rates, typically hundreds of nanoliters / minute of sample solution or lower without the use of external solvent delivery. The electrospray infusion device that forms the nano-electrospray can use a static nano-electrospray emitter or a dynamic nano-electrospray emitter. The static nano-electrospray emitter performs continuous analysis of a small sample (analyte) solution volume over an extended period of time. Before being analyzed by a mass spectrometer, a dynamic nano-electrospray emitter uses a capillary column and a solvent delivery system to chromatographically separate the mixture.

[0152] In some exemplary embodiments, mass spectrometry can be performed under native conditions. As used herein, the term "native conditions" may include performing mass spectrometry under conditions that retain non-covalent interactions in the analyte. For a detailed review of native MS, see the following review: Elisabetta Boeri Erba and Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE 1176-1192 (2015).

[0153] As used herein, the term "database" refers to a collection of protein sequences that may be present in a sample, for example, in the form of a file in FASTA format. The relevant protein sequence may be derived from the cDNA sequence of the species being studied. Public databases that can be used to search for relevant protein sequences include databases hosted by, for example, Uniprot or Swiss-prot. The database can be searched using a tool referred to herein as a "bioinformatic tool". The bioinformatic tool provides the ability to search for uninterpreted MS / MS spectra relative to all possible sequences in the database, and provides interpreted (annotated) MS / MS spectra as output. Non-limiting examples of such tools are Mascot (www.matrixscience.com), Spectrum Mill (www.chem.agilent.com), PLGS (www.waters.com), PEAKS (www.bioinformaticssolutions.com), Proteinpilot (download.appliedbiosystems.com / proteinpilot), Phenyx (www.phenyx-ms.com), Sorcerer (www.sagenresearch.com), OMSSA (www.pubchem.ncbi.nlm.nih.gov / omssa / ), X! Tandem (www.thegpm.org / TANDEM / ), Protein Prospector (prospector.ucsf.edu / prospector / mshome.htm), Byonic (www.proteinmetrics.com / products / byonic), or Sequest (fields.scripps.edu / sequest).

[0154] As used herein, the term "downstream process technology" refers to one or more technologies used after upstream process technologies for producing, treating and / or purifying proteins. For example, downstream process technologies include separation of protein products using, for example, affinity chromatography, ion exchange chromatography (such as anion or cation exchange chromatography), hydrophobic interaction chromatography, or displacement chromatography.

[0155] As used herein, the term "ultrafiltration" or "UF" may include a membrane filtration process similar to reverse osmosis, using hydrostatic pressure to force water through a semipermeable membrane. Ultrafiltration is described in detail in: LEOS J. ZEMAN and Andrew L. ZYDNEY, MICROFILTRATION AND ULTRAFILTRATION: PRINCIPLES AND APPLICATIONS (1996), the entire teaching of which is incorporated herein. Filters with a pore size of less than 0.1 μm can be used for ultrafiltration. By using a filter with such a small pore size, the sample volume can be reduced by allowing the sample buffer to permeate through the filter while retaining proteins behind the filter.

[0156] As used herein, "diafiltration" or "DF" may include methods of using an ultrafilter to remove and exchange salts, sugars, and non-aqueous solvents, thereby separating free species from bound species, removing low molecular weight substances, and / or causing changes in the ionic and / or pH environment. Trace solutes can be most effectively removed by adding solvent to the solution being ultrafiltered at a rate approximately equal to the ultrafiltration rate. This washes trace species from the solution at a constant volume. In certain exemplary embodiments of the present disclosure, the diafiltration step can be used, for example, to exchange various buffers used in conjunction with the present disclosure prior to chromatography or other production steps, as well as to remove impurities from protein preparations.

[0157] As used herein, the term "formulation" refers to a pharmaceutical product, such as dupilumab, formulated together with one or more pharmaceutically acceptable vehicles.

[0158] With respect to protein formulations, the term "stable" as used herein means that the protein of interest within the formulation is able to retain an acceptable degree of chemical structure or biological function after storage under the exemplary conditions defined herein. A formulation may be stable even if the protein of interest contained therein does not maintain 100% of its chemical structure or biological function after storage for a specified amount of time. In some cases, maintaining about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure or function of the protein after storage for a specified amount of time may be considered "stable."

[0159] The terms "treat" or "treatment" refer to therapeutic measures to reverse, stabilize or eliminate an undesirable disease or condition (e.g., atopic dermatitis (eczema), eosinophilic or oral steroid-dependent asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), eosinophilic esophagitis (EoE), inflammation), for example, by causing regression, stabilization or elimination of one or more symptoms or hallmarks of such disease or condition to any clinically measurable extent, e.g., for asthma, it helps prevent severe asthma attacks (exacerbations), can improve breathing, and helps reduce the amount of oral corticosteroids required.

[0160] As used herein, the term "critical quality attribute" (CQA) is used to describe a physical, chemical, biological or microbiological property or characteristic that should be within an appropriate limit, range or distribution to ensure the desired product quality. CQAs may include, for example, post-translational modifications.

[0161] As used herein, "virus filtration" may include filtering using a suitable filter, including but not limited to Planova 20N from Asahi Kasei Pharma. TM , 50N or BioEx, Viresolve from EMD Millipore TM Filters from Sartorius CPV or HF, or Ultipor DV20 or DV50 from Pall Corporation TM or Pegasus TM Prime Filter. It will be apparent to one of ordinary skill in the art that an appropriate filter is selected to obtain the desired filtration performance.

[0162] For biologics, the implementation of a robust and flexible upstream process is desirable. An efficient upstream process can result in the desired production and scale-up of the protein of interest.

[0163] As used herein, a "sample" can be obtained from any step in a bioprocess, such as a cell culture fluid (CCF), a collected cell culture fluid (HCCF), any step in a downstream process, a drug substance (DS), or a drug product (DP) comprising a final formulated product. In some specific exemplary embodiments, the sample can be selected from any step in a downstream process of clarification, chromatography production, or filtration.

[0164] As used herein, the term "protein alkylating agent" or "alkylation agent" refers to an agent used to alkylate certain free amino acid residues in proteins. Non-limiting examples of protein alkylating agents are iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine, or a combination thereof.

[0165] As used herein, "denaturing" or "denaturation" may refer to a process in which the three-dimensional shape of a molecule is changed from its native state. Protein denaturation may be performed using a protein denaturant. Non-limiting examples of protein denaturants include heat, high or low pH, reducing agents (such as DTT, SDS), or exposure to a chaotropic agent. Several chaotropic agents may be used as protein denaturants. Chaotropic solutes increase the entropy of a system by interfering with intramolecular interactions mediated by non-covalent forces (such as hydrogen bonds, van der Waals forces, and hydrophobic interactions). Non-limiting examples of chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate (SDS), thiourea, N-lauroyl sarcosine, urea, and salts thereof. In some exemplary embodiments, a denaturant may be used in a mobile phase in a chromatographic analysis. In some exemplary embodiments, the denaturant used in the mobile phase may be acetonitrile (ACN). In some exemplary embodiments, the denaturant used in the mobile phase may be a surfactant.

[0166] As used herein, the term "digestion" refers to the hydrolysis of one or more peptide bonds of a protein. There are several methods for digesting proteins in a sample using appropriate hydrolyzing agents, such as enzymatic digestion or non-enzymatic digestion. Digestion of proteins into peptides can produce "peptide digests" that can be further analyzed using peptide mapping analysis.

[0167] As used herein, the term "hydrolyzing agent" refers to any one or a combination of many different agents that can perform protein digestion. Non-limiting examples of hydrolyzing agents that can perform enzymatic digestion include proteases from Aspergillus saitoi, elastase, subtilisin, proteinase XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillus pepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C) or outer membrane protein T (OmpT), immunoglobulin degrading enzyme (IdeS) of Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease or biologically active fragments or homologs thereof or combinations thereof. Non-limiting examples of hydrolytic agents that can perform non-enzymatic digestion include the use of high temperature, microwaves, ultrasound, high pressure, infrared, solvents (non-limiting examples are ethanol and acetonitrile), immobilized enzyme digestion (IMER), magnetic particle immobilized enzymes, and on-chip immobilized enzymes. For a recent review of available techniques for protein digestion, see Switzar et al., "Protein Digestion: An Overview of the Available Techniques and Recent Developments" (Linda Switzar, Martin Giera, and Wilfried MANiessen, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 Journal of Proteome Research 1067-1077 (2013), all of which are incorporated herein). One or a combination of hydrolytic agents can cleave peptide bonds in proteins or polypeptides in a sequence-specific manner, thereby generating a predictable set of shorter peptides. The ratio of hydrolytic agent to protein and the time required for digestion can be appropriately selected to obtain optimal digestion of protein. When the enzyme: substrate ratio is inappropriately high, the corresponding high digestion rate will not allow the mass spectrometer enough time to analyze the peptides, and sequence coverage will be compromised. On the other hand, a low E / S ratio will require a longer time to digest and therefore a longer data acquisition time. The enzyme: substrate ratio can be in the range of about 1:0.5 to about 1:200.

[0168] As used herein, the term "protein reducing agent" or "reduction agent" refers to an agent used to reduce disulfide bridges in proteins. Non-limiting examples of protein reducing agents used to reduce proteins are dithiothreitol (DTT), β-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl) or a combination thereof.

[0169] As used herein, the term "protein" or "protein of interest" may include any amino acid polymer with covalently linked amide bonds. Proteins include one or more amino acid polymer chains, which are generally referred to as "polypeptides" in the art. "Polypeptide" refers to a polymer consisting of amino acid residues, their naturally occurring related structural variants, and non-naturally occurring synthetic analogs connected via peptide bonds. "Synthetic peptides or polypeptides" refer to non-naturally occurring peptides or polypeptides. Synthetic peptides or polypeptides can be synthesized, for example, using an automated peptide synthesizer. Various solid-phase peptide synthesis methods are known to those skilled in the art. A protein may include one or more polypeptides to form a single functional biomolecule. In another exemplary aspect, a protein may include an antibody fragment, a nanobody, a recombinant antibody chimera, a cytokine, a chemokine, a peptide hormone, etc. The protein of interest may include any of a biotherapeutic protein, a recombinant protein for research or therapy, a chimeric protein, an antibody, a monoclonal antibody, a polyclonal antibody, and a human antibody. Proteins can be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia species), and mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation" (Darius Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012), the entire teachings of which are incorporated herein by reference). In some exemplary embodiments, the protein comprises modifications, adducts, and other covalently linked moieties. These modifications, adducts and moieties include, for example, avidin, streptavidin, biotin, polysaccharides (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose and other monosaccharides), PEG, polyhistidine, FLAG tags, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST), myc-epitopes, fluorescent labels and other dyes, etc.Proteins can be classified based on composition and solubility and can therefore include simple proteins, such as globular proteins and fibrous proteins; bound proteins, such as nucleoproteins, glycoproteins, mucins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins.

[0170] As used herein, the term "recombinant protein" refers to a protein produced by transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein may be an antibody, such as a chimeric antibody, a humanized antibody, or a fully human antibody. In certain exemplary embodiments, the recombinant protein may be an antibody of an isotype selected from the group consisting of IgG, IgM, IgA1, IgA2, IgD, and IgE. In certain exemplary embodiments, the antibody molecule is a full-length antibody (e.g., IgG1), or alternatively, the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0171] As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In various embodiments of the present disclosure, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be consistent with human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. As used herein, the term "antibody" also includes antigen-binding fragments of intact antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from, for example, intact antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optional constant domains. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example to arrange one or more variable domains and / or constant domains into a suitable configuration, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0172] As used herein, "antibody fragments" include a portion of an intact antibody, such as an antigen binding region or variable region of an antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv bifunctional antibodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementary determining regions (CDRs), as well as trifunctional antibodies, tetrafunctional antibodies, linear antibodies, and single-chain antibody molecules. Fv fragments are a combination of the variable regions of immunoglobulin heavy and light chains, and ScFv proteins are recombinant single-chain polypeptide molecules in which the immunoglobulin light and heavy chain variable regions are connected by a peptide linker. In some exemplary embodiments, an antibody fragment comprises sufficient amino acid sequence of a parent antibody to make it a fragment that binds to the same antigen as the parent antibody; in some exemplary embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Antibody fragments may be produced by any means. For example, antibody fragments can be produced enzymatically or chemically by fragmentation of intact antibodies and / or they can be produced recombinantly from genes encoding partial antibody sequences. Alternatively or additionally, antibody fragments can be produced in whole or in part synthetically. Antibody fragments can optionally comprise single-chain antibody fragments. Alternatively or additionally, antibody fragments can comprise multiple chains linked together, for example, by disulfide bonds. Antibody fragments can optionally comprise multimolecular complexes. Functional antibody fragments typically comprise at least about 50 amino acids and more typically comprise at least about 200 amino acids.

[0173] As used herein, the term "monoclonal antibody" is not limited to antibodies produced via fusion tumor technology. Monoclonal antibodies can be derived from a single strain by any means available or known in the art, including any eukaryotic, prokaryotic or phage strain. Monoclonal antibodies useful in the present disclosure can be prepared using a wide variety of techniques known in the art, including the use of fusion tumors, recombination, and phage display technology, or a combination thereof.

[0174] As used herein, "protein pharmaceutical products," "biopharmaceutical products," or "biotherapeutics" include active ingredients that may be wholly or partially biological in nature. In one aspect, a protein pharmaceutical product may comprise a peptide, protein, antibody, antigen, peptide-drug conjugate, antibody-drug conjugate, protein-drug conjugate, cell, tissue, or a combination thereof. In another aspect, a protein pharmaceutical product may comprise a recombinant, engineered, modified, mutated, or truncated form of a peptide, protein, antibody, antigen, vaccine, peptide-drug conjugate, antibody-drug conjugate, protein-drug conjugate, cell, tissue, or a combination thereof.

[0175] Exemplary Anti-IL-4Rα Antibodies

[0176] As used herein, dupilumab (IgG4 isotype) is a covalent heterotetramer composed of two disulfide-linked human heavy chains, each covalently linked to a human kappa light chain via a disulfide bond. Each heavy chain contains a serine to proline mutation at amino acid 233 located in the hinge region of the Fc domain to reduce the tendency of the antibody to form half antibodies in solution. There is a single N-linked glycosylation site (Asn 302 ), which is located in the CH2 domain of the Fc constant region of the molecule. The antibody based on the primary sequence (without N-linked glycosylation) has a molecular weight of 146,897.0 Da, considering the formation of 16 disulfide bonds and the removal of Lys from the C-terminus of each heavy chain. 452 The variable domains of the heavy and light chains combine to form the complementarity determining regions (CDRs) that are used to bind dupilumab to its target, interleukin-4 receptor alpha (IL-4Rα). Figure 1 A schematic representation of dupilumab, including the location of N-linked glycosylation sites and disulfide bond structure, is presented in FIG.

[0177] The manufacturing process can be used to produce a human antibody or antigen-binding fragment thereof that specifically binds to an IL-4R (e.g., IL-4Rα) and comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 1. The antibody or antigen-binding fragment may comprise three light chain CDRs (LCVR1, LCVR2, LCVR3) contained within a light chain variable region (LCVR) having an amino acid sequence of SEQ ID NO: 2. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within a given HCVR and / or LCVR amino acid sequence disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general, the Kabat definition is based on sequence variability, the Chothia definition is based on the position of the structural loop region, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases can also be used to identify CDR sequences within antibodies.

[0178] In certain embodiments, the antibody or antigen-binding fragment thereof comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) from the heavy chain and light chain variable region amino acid sequence pair (HCVR / LCVR) of SEQ ID NOs: 1 and 2.

[0179] In certain embodiments, the antibody or antigen-binding fragment thereof comprises six CDRs (HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3) having the amino acid sequence of SEQ ID NO: 3 / 4 / 5 / 6 / 7 / 8.

[0180] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 1 and 2.

[0181] In certain embodiments, the antibody is dupilumab, which comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 1 and 2.

[0182] In certain embodiments, the antibody sequence is dupilumab, which comprises the heavy chain / light chain amino acid sequence pair of SEQ ID NOs: 9 and 10.

[0183] Dupilumab HCVR amino acid sequence:

[0184] EVQLVESGGGLEQPGGSLRLSCAGSGFFTFRDYAMTWVRQAPGKGLEWVSSISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRLSITIRPRYYGLDV WGQGTTVTVS (SEQ ID NO: 1).

[0185] Dupilumab LCVR amino acid sequence:

[0186] DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLLIYLGSN RASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTPYTFGQGTKLEIK (SEQ ID NO: 2).

[0187] Dupilumab HCDR1 amino acid sequence:

[0188] GFTFRDYA (SEQ ID NO: 3).

[0189] Dupilumab HCDR2 amino acid sequence:

[0190] ISGSGGNT (SEQ ID NO: 4).

[0191] Dupilumab HCDR3 amino acid sequence:

[0192] AKDRLSITIRPRYYGL (SEQ ID NO: 5).

[0193] Dupilumab LCDR1 amino acid sequence:

[0194] QSLLYSIGYNY (SEQ ID NO: 6).

[0195] Dupilumab LCDR2 amino acid sequence:

[0196] LGS (SEQ ID NO: 7).

[0197] Dupilumab LCDR3 amino acid sequence:

[0198] MQALQTPYT (SEQ ID NO:8).

[0199] Dupilumab HC amino acid sequence:

[0200] EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVSSISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRLSITIRPRYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:9) (amino acids 1 - 124 = HCVR; amino acids 125 - 451 = HC constant region).

[0201] Dupilumab LC amino acid sequence:

[0202] DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:10) (amino acids 1 - 112 = LCVR; amino acids 112 - 219 = LC constant region).

[0203] In certain embodiments, the manufacturing method can be used to produce an antibody or antigen-binding fragment thereof, which comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR) sequence pair (LCVR / HCVR) selected from the group consisting of SCB-VL-39 / SCB-VH-92; SCB-VL-40 / SCB-VH-92; SCB-VL-41 / SCB-VH-92; SCB-VL-42 / SCB-VH-92; SCB-VL-43 / SCB-VH-92; SCB-VL-44 / SCB-VH-92; SCB-VL-44 / SCB-VH-62; SCB-VL-44 / SCB-VH-68; SCB-VL-44 / SCB-VH-70; SCB-VL-45 / SCB-VH-71; SCB-VL-46 / SCB-VH-72; SCB-VL-47 / SCB-VH-73; SCB-VL-48 / SCB-VH-74. B-VH-72;SCB-VL-44 / SCB-VH-82;SCB-VL-44 / SCB-VH-85;SCB-VL-44 / SCB-VH-91; SCB-VL-44 / SCB-VH-93; -92;SCB-VL-47 / SCB-VH-92;SCB-VL-48 / SCB-VH-92;SCB-VL-49 / SCB-VH-92;SCB-VL-50 / SCB-VH-92; SCB-VL-51 / SCB-VH-92; CB-VL-52 / SCB-VH-92;SCB-VL-52 / SCB-VH-62;SCB-VL-52 / SCB-VH-91;SCB-VL-53 / SCB-VH-92;SCB-VL-54 / SCB-VH-92;SCB-VL-54 / SCB-VH-62;SCB-V SCB-VL-54 / SCB-VH-91; SCB-VL-55 / SCB-VH-92; SCB-VL-55 / SCB-VH-62; SCB-VL-55 / SCB-VH-68; SCB-VL-55 / SCB-VH-72; SCB-VL-55 / SCB-VH-82; H-92;SCB-VL-57 / SCB-VH-92;SCB-VL-57 / SCB-VH-93;SCB-VL-57 / SCB-VH-59;SCB-VL-57 / SCB-VH-60;SCB-VL-57 / SCB-VH-63; SCB-VL-57 / SCB-VH-64; SCB-VL-57 / SCB-VH-65; SCB-VL-57 / SCB-VH-66; SCB-VL-57 / SCB-VH-67; SCB-VL-57 / SCB-VH-68; SCB-VL-57 / SCB-VH-69; SCB-VL-57 / SCB-VH-70; SCB-VL-57 / SCB-VH-71; SCB-VL-57 / SCB-VH-72; SCB-VL-57 / SCB-VH-73; SCB-VL-57 / SCB-VH-74; SCB-VL-57 / SCB-VH-75; SCB-VL-57 / SCB-VH-76; SCB-VL-57 / SCB-VH-77; SCB-VL-57 / SCB-VH-78; SCB-VL-57 / SCB-VH-79; SCB-VL-57 / SCB-VH-80; SCB-VL-57 / SCB-VH-81; SCB-VL-57 / SCB-VH-82; SCB-VL-57 / SCB-VH-83; SCB-VL-57 / SCB-VH-84; SCB-VL-57 / SCB-VH-85; SCB-VL-57 / SCB-VH-86; SCB-VL-57 / SCB-VH-87; SCB-VL-57 / SCB-VH-88; SCB-VL-57 / SCB-VH-89; SCB-VL-57 / SCB-VH-90; SCB-VL-57 / SCB-VH-91; SCB-VL-58 / SCB-VH-91; SCB-VL-58 / SCB-VH-92; and SCB-VL-58 / SCB-VH-93. ;

[0204] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the LCVR / HCVR sequence pair of SCB-VL-44 / SCB-VH-92.

[0205] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the LCVR / HCVR sequence pair of SCB-VL-54 / SCB-VH-92.

[0206] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the LCVR / HCVR sequence pair of SCB-VL-55 / SCB-VH-92.

[0207] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a HCVR comprising the HCDR1 sequence of SCB-92-HCDR1, the HCDR2 sequence of SCB-92-HCDR2, and the HCDR3 sequence of SCB-92-HCDR3; and a LCVR comprising the LCDR1 sequence of SCB-55-LCDR1, the LCDR2 sequence of SCB-55-LCDR2, and the LCDR3 sequence of SCB-55-LCDR3.

[0208] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a HCVR comprising the HCDR1 sequence of SCB-92-HCDR1, the HCDR2 sequence of SCB-92-HCDR2, and the HCDR3 sequence of SCB-92-HCDR3; and a LCVR comprising the LCDR1 sequence of SCB-55-LCDR1, the LCDR2 sequence of SCB-54-LCDR2, and the LCDR3 sequence of SCB-55-LCDR3.

[0209] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a HCVR comprising the HCDR1 sequence of SCB-92-HCDR1, the HCDR2 sequence of SCB-92-HCDR2, and the HCDR3 sequence of SCB-92-HCDR3; and a LCVR comprising the LCDR1 sequence of SCB-55-LCDR1, the LCDR2 sequence of SCB-54-LCDR2, and the LCDR3 sequence of SCB-44-LCDR3.

[0210] The antibodies listed in Table 1 below are described in more detail in U.S. Patent No. 10,774,141, which is incorporated herein by reference in its entirety for all purposes.

[0211] Table 1

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] In certain embodiments, the manufacturing method can be used to produce an antibody or antigen-binding fragment thereof, which comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR) sequence pair (LCVR / HCVR) selected from the group consisting of MEDI-1-VL / MEDI-1-VH to MEDI-42-VL / MEDI-42-VH.

[0218] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the LCVR / HCVR sequence pair of MEDI-37GL-VL / MEDI-37GL-VH.

[0219] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a HCVR comprising the HCDR1 sequence of MEDI-37GL-HCDR1, the HCDR2 sequence of MEDI-37GL-HCDR2, and the HCDR3 sequence of MEDI-37GL-HCDR3; and a LCVR comprising the LCDR1 sequence of MEDI-37GL-LCDR1, the LCDR2 sequence of MEDI-37GL-LCDR2, and the LCDR3 sequence of MEDI-37GL-LCDR3.

[0220] The antibodies listed in Table 2 below are described in more detail in U.S. Patent No. 8,877,189, which is incorporated herein by reference in its entirety for all purposes.

[0221] Table 2

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the LCVR / HCVR sequence pair of AJOU-90-VL / AJOU-83-VH.

[0231] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a HCVR comprising the HCDR1 sequence of AJOU-84-HCDR1, the HCDR2 sequence of AJOU-85-HCDR2, and the HCDR3 sequence of AJOU-32-HCDR3; and a LCVR comprising the LCDR1 sequence of AJOU-96-LCDR1, the LCDR2 sequence of AJOU-60-LCDR2, and the LCDR3 sequence of AJOU-68-LCDR3.

[0232] The antibodies listed in Table 3 below are described in more detail in WO2020 / 096381 and Kim et al. (Scientific Reports. 9:7772.2019), which are incorporated herein by reference in their entirety for all purposes.

[0233] Table 3

[0234]

[0235]

[0236]

[0237]

[0238] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a light chain variable region (LCVR) and a heavy chain variable region (HCVR) sequence pair (LCVR / HCVR) selected from the group of sequences listed in Table 4 consisting of 11 / 3, 27 / 19, 43 / 35, 59 / 51, 75 / 67, 91 / 83, 107 / 99, 123 / 115, 155 / 147 and 171 / 163.

[0239] The antibodies listed in Table 4 below are described in more detail in U.S. Patent No. 7,605,237 and U.S. Patent No. 7,608,693, which are incorporated herein by reference in their entirety for all purposes.

[0240] Table 4

[0241]

[0242]

[0243]

[0244] It will be understood that the present disclosure is not limited to any of the aforementioned proteins, proteins of interest, antibodies, cell culture media, host cell proteins, protein alkylating agents, protein denaturing agents, protein reducing agents, digestive enzymes, treatments, processes, samples, surfactants, detergents, chromatography methods, filtration methods, mass spectrometers, databases, bioinformatics tools, pH, temperature, or concentrations, and that any protein, protein of interest, antibody, cell culture media, host cell proteins, protein alkylating agents, protein denaturing agents, protein reducing agents, digestive enzymes, treatments, processes, samples, surfactants, detergents, chromatography methods, filtration methods, mass spectrometers, databases, bioinformatics tools, pH, temperature, or concentration may be selected by any suitable means.

[0245] The present disclosure will be more fully understood with reference to the following examples. However, the examples should not be construed as limiting the scope of the invention.

[0246] Cell culture media and supplements

[0247] Unfortunately, removing serum and reducing or eliminating hydrolysates from cell culture media while reducing batch-to-batch variability and facilitating downstream processing steps can reduce cell growth, reduce viability, and reduce protein expression. Therefore, chemically defined serum-free and low-content or even hydrolysate-free culture media require additional ingredients to improve cell growth and protein production. The cell culture media of the present invention can be supplemented with additional ingredients, such as polyamines, or with increased concentrations of components, such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, etc., depending on the needs of the cells to be cultured or the desired cell culture parameters.

[0248] In some embodiments, the cell culture medium disclosed herein is supplemented with polyamines such as ornithine, putrescine, spermine, spermidine, or a combination thereof ("PS medium") to improve cell growth, cell viability, and recombinant protein production.

[0249] In one aspect, the PS medium comprises a concentration (expressed in micromoles / liter) of at least about 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 540, 545, 550, 555, 560, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 800, 850, or 900 ± 14 μM ornithine. In one aspect, the PS medium comprises ornithine at a concentration between about 0.09 and 0.9 mM.

[0250] In one embodiment and in addition to including ornithine or putrescine, the culture medium comprises a mixture of nucleosides at a cumulative concentration of at least 50 μM, at least 60 μM, at least 70 μM, at least 80 μM, at least 90 μM, at least 100 μM, at least 110 μM, at least 115 μM, at least 120 μM, at least 125 μM, at least 130 μM, at least 135 μM, at least 140 μM, at least 145 μM, at least 150 μM, at least 155 μM, at least 160 μM, at least 165 μM, or at least 170 μM.

[0251] In one embodiment, the culture medium comprises about 174 μM ± 26 μM nucleosides. In one embodiment, the culture medium comprises a cumulative concentration of at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, at least 65 μM, at least 70 μM, at least 75 μM, at least 80 μM, at least 85 μM, at least 90 μM, at least 95 μM, at least 100 μM or at least 105 μM purine derivatives. In one embodiment, the culture medium comprises about 106 μM ± 5 μM purine derivatives. Purine derivatives include hypoxanthine and nucleosides adenosine and guanosine. In one embodiment, the culture medium comprises a cumulative concentration of at least 30 μM, at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM or at least 65 μM pyrimidine derivatives. In one embodiment, the culture medium comprises about 68 μM ± 5 μM pyrimidine derivatives. Pyrimidine derivatives include the nucleosides thymidine, uridine and cytidine. In a specific embodiment, the culture medium comprises adenosine, guanosine, cytidine, uridine, thymidine and hypoxanthine.

[0252] In one embodiment, in addition to ornithine or putrescine, the culture medium further comprises an amino acid at a cumulative concentration of at least 40 mM, wherein the amount of glutamine is not included in the calculation of the cumulative total amount. In one embodiment, glutamine is not included in the culture medium, but can be supplied to the culture medium as a "point of use addition" during the cultivation of cells, such as during the production of proteins. Therefore, in some embodiments, such as in methods of culturing cells or methods of producing proteins of interest, the culture medium can be supplemented with glutamine as a point of use addition. In one such embodiment, glutamine is added in an amount of less than about 40 mM, less than about 35 mM, less than about 30 mM, less than about 25 mM, less than about 20 mM, less than about 15 mM, less than about 10 mM, less than about 8 mM, less than about 7 mM, less than about 6 mM, less than about 5 mM, less than about 4 mM, less than about 3 mM, or less than about 2.5 mM. In one embodiment, the amount of glutamine in the culture medium supplemented with glutamine is about 2 mM ± 0.5 mM.

[0253] In one embodiment, in addition to including ornithine or a combination of ornithine and putrescine, the culture medium also comprises an amino acid with a non-polar side group at a concentration of at least 15mM, at least 24mM, at least 25mM, at least 26mM, at least 27mM, at least 28mM, at least 29mM or at least 30mM. In one embodiment, the culture medium comprises about 30mM amino acids with non-polar side groups. In one embodiment, of the total amount of amino acids contained in the culture medium by mole, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40% or at least 41% are amino acids with non-polar side groups. In one embodiment, about 42 mol % ± 1 mol % of the amino acids in the culture medium are amino acids with non-polar side groups. Amino acids with non-polar side groups include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine. In one embodiment, the optional amino acid mixture supplement is selected from the group consisting of the amino acids in Table 5 below:

[0254] Table 5

[0255] Amino Acids Range mM (mmol / L) Range (g / L) Alanine 0-11.2 0-1 Arginine 2.4-11.9 0.5-2.5 Asparagine 1.3-33.3 0.2-5 Aspartic acid 1.5-93.9 0.2-12.5 Cysteine 1.1-19.9 0.2-3.5 Glutamate 1.4-47.6 0.2-7 Glutamine 0-23.9 0-3.5 Glycine 0-16.7 0-1.25 Histidine 1-9.5 0.2-2 Isoleucine 1.5-22.9 0.2-3 Leucine 1.5-38.1 0.2-5 Lysine 2.7-24.6 0.5-4.5 Methionine 1.3-13.4 0.2-2 Phenylalanine 1.2-18.2 0.2-3 Proline 1.7-26.1 0.2-3 Serine 1.9-57.1 0.2-6 Threonine 1.7-33.6 0.2-4 Tryptophan 0.5-14.7 0.1-3 Tyrosine 0.9-22.2 0.2-5 Valine 1.7-34.1 0.2-4

[0256] In one embodiment, in addition to including ornithine or a combination of ornithine and putrescine, the culture medium also comprises an amino acid with uncharged polar side groups at a concentration of about 10mM to 34mM, about 11mM to 33mM, about 12mM to 32mM, about 13mM to 31mM, about 14mM to 30mM, about 15mM to 29mM, about 16mM to 28mM, about 17mM to 27mM, about 18mM to 26mM, about 19mM to 25mM, about 20mM to 24mM, about 21mM to 23mM, about 22mM, about 23mM, about 24mM, or about 25mM. In one embodiment, the culture medium comprises about 22mM of amino acids with uncharged polar side groups. In another embodiment, the culture medium comprises about 12mM of amino acids with uncharged polar side groups. In one embodiment, in the total amount of amino acids by mole contained in the culture medium, about 14% to 46%, about 15% to 45%, about 16% to 44%, about 17% to 43%, about 18% to 42%, about 19% to 41%, about 20% to 40%, about 21% to 39%, about 22% to 38%, about 23% to 37%, about 24% to 36%, about 25% to 35%, about 26% to 34%, about 27% to 33%, about 28% to 32%, about 29% to 31%, or about 30% are amino acids with uncharged polar side groups. In one embodiment, about 30 mol% ± 3 mol% of amino acids in the culture medium are amino acids with uncharged polar side groups. Amino acids with uncharged polar side groups include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.

[0257] In one embodiment, in addition to including ornithine or a combination of ornithine and putrescine, the culture medium also includes an amino acid (e.g., an acidic amino acid) having a negative charge at pH 6 at a concentration of about 4mM to 14mM, about 5mM to 13mM, about 6mM to 12mM, about 7mM to 11mM, about 8mM to 10mM, about 9mM, or about 4mM. In one embodiment, the culture medium includes about 9mM of acidic amino acids. In one embodiment, the culture medium includes 9mM ± 1mM of acidic amino acids. In one embodiment, of the total amount of amino acids contained in the culture medium by mole, about 8% to 18%, about 9% to 17%, about 10% to 16%, about 11% to 15%, about 12% to 14%, or about 13% are acidic amino acids. In one embodiment, about 12.6 mol% ± 1 mol% of the amino acids in the culture medium are acidic amino acids. Acidic amino acids include aspartic acid and glutamic acid.

[0258] In one embodiment, in addition to ornithine or a combination of ornithine and putrescine, the culture medium also comprises a concentration of at least 3.5 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, or at least 11 mM of an amino acid (e.g., a basic amino acid) having a positive charge at pH 6. In one embodiment, the culture medium comprises about 11 mM of basic amino acids. In one embodiment, the culture medium comprises about 11.42 mM ± 1 mM of basic amino acids. In one embodiment, of the total amount of amino acids contained in the culture medium by mole, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15% are basic amino acids. In one embodiment, about 16 mol% of the amino acids in the culture medium are basic amino acids. In one embodiment, about 15.8 mol% ± 2.4 mol% of the amino acids in the culture medium are basic amino acids. In one embodiment, about 21 mol% ± 3.2 mol% of the amino acids in the culture medium are basic amino acids. Basic amino acids include lysine, arginine and histidine.

[0259] In one embodiment, in addition to including ornithine or a combination of ornithine and putrescine, the culture medium also comprises about 30 mM nonpolar amino acids, about 22 mM uncharged polar amino acids, about 9 mM acidic amino acids, and about 11 mM basic amino acids. In one embodiment, of the amino acids in the culture medium, about 42 mole % are nonpolar amino acids, about 30 mole % are uncharged polar amino acids, about 13 mole % are acidic amino acids, and about 16 mole % are basic amino acids.

[0260] In one embodiment, in addition to comprising ornithine or a combination of ornithine and putrescine, the culture medium also comprises micromolar amounts of fatty acids (or fatty acid derivatives) and tocopherol. In one embodiment, the fatty acids comprise any one or more of the following: linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, capric acid, dodecanoic acid, caproic acid, lignoceric acid, myristic acid and caprylic acid. In one embodiment, the culture medium comprises tocopherol, linoleic acid and thioctic acid.

[0261] In one embodiment, the culture medium also includes a mixture of vitamins having a cumulative concentration of at least about 700 μM or at least about 2 mM, including other nutrients and essential nutrients. In one embodiment, the mixture of vitamins includes one or more of: D-biotin, choline chloride, folic acid, inositol, nicotinamide, pyridoxine HCl, D-pantothenic acid (half calcium), riboflavin, thiamine HCl, vitamin B12, etc. In one embodiment, the mixture of vitamins includes all of D-biotin, choline chloride, folic acid, inositol, nicotinamide, pyridoxine HCl, D-pantothenic acid (half calcium), riboflavin, thiamine HCl, and vitamin B12.

[0262] In one embodiment, the culture medium further comprises taurine, hypotaurine or a combination thereof. Taurine is present in many tissues of humans and other mammalian species, such as brain, retina, myocardium, skeleton and smooth muscle, platelets and neutrophils. Taurine can contribute to osmotic pressure regulation, membrane stability and anti-inflammatory, and regulates mitochondrial protein synthesis (Jong et al., 2010, Journal of Biomedical Science 17 (Supplement 1) via the enhanced electron transport chain activity that avoids superoxide production (Jong et al., 2010, Journal of Biomedical Science 17 (Supplement 1): S25; Jong et al., 2012, Amino Acids 42: 2223-2232sw). In one aspect, the cell culture medium without serum may comprise about 0.1mM to about 10mM taurine, about 0.1mM to about 1mM taurine, about 0.4mM to about 8mM taurine, about 1mM to about 6mM taurine, about 2mM to about 5mM taurine or about 3 to about 4mM taurine. It has also been found that taurine increases the specific productivity of cells and enables those cells to produce less ammonia byproducts, thereby improving the production of recombinant proteins. See U.S. Patent No. 10,927,342, which is incorporated by reference in its entirety.

[0263] In one aspect, a fed-batch production method for preparing dupilumab comprises culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding said dupilumab on a large scale in a cell culture production medium, wherein insulin in the medium is supplemented on days 2 and 4 at a concentration of about 7.5 mg / L.

[0264] In one embodiment, the dupilumab is produced in the cell culture production medium at a titer of at least 1.5 g / L on day 4. In another embodiment, the dupilumab is produced in the cell culture production medium at a titer of at least 2 g / L on day 4.

[0265] In one embodiment, the culturing step lasts for about 10 to 18 days. In another embodiment, the culturing step lasts for about 10 days, about 12 days, about 14 days, about 16 days, or about 18 days.

[0266] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium comprises a recombinant growth factor, an osmotic weight molar concentration regulator, a pH buffer, glutamine and a cell protectant.

[0267] In one embodiment, the CHO cells are cultured at a temperature in the range of about 32°C to about 38°C.

[0268] In one embodiment, the dupilumab is produced in the cell culture production medium at a titer of at least about 0.5 g / L on day 4. In another embodiment, the dupilumab is produced such that the viability of the cells in the cell culture production medium is at least about 95% on day 4. In yet another embodiment, the dupilumab is produced such that the ammonia concentration in the cell culture production medium on day 4 is less than about 5 mM.

[0269] In one aspect, a fed-batch production method for preparing dupilumab comprises culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding said dupilumab at large scale in a cell culture production medium, wherein tyrosine in said medium is supplemented at a concentration of about 2 g / L on day 3.

[0270] In one embodiment, the dupilumab is produced in the cell culture production medium at a titer of at least about 8 g / L on day 14.

[0271] In one embodiment, the dupilumab is produced in the cell culture production medium at a titer of at least 9 g / L on day 10 to 14. In another embodiment, the dupilumab is produced in the cell culture production medium at a titer of at least 10 g / L on day 10 to 14.

[0272] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium comprises a recombinant growth factor, an osmotic weight molar concentration regulator, a pH buffer, glutamine, methotrexate, and a cell protectant.

[0273] In one embodiment, the CHO cells are cultured at a temperature in the range of about 32°C to about 38°C.

[0274] In one embodiment, the ammonia concentration in the cell culture production medium is less than about 10 mM on day 14. In another embodiment, the ammonia concentration in the cell culture production medium is less than about 8 mM on day 14.

[0275] In one aspect, a fed-batch production method for preparing dupilumab comprises culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding said dupilumab at large scale in a cell culture production medium, wherein tyrosine in said medium is supplemented at a concentration of about 2 g / L on days 3 and 7.

[0276] In one aspect, a fed-batch production method for preparing dupilumab comprises culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding said dupilumab on a large scale in a cell culture production medium, wherein tyrosine in the medium is supplemented at a concentration of about 1 g / L on days 3 and 7.

[0277] In one embodiment, the dupilumab is produced in the cell culture production medium at a titer of at least about 8 g / L on day 14. In another embodiment, the dupilumab is produced in the cell culture production medium at a titer of at least 9 g / L. In yet another embodiment, the dupilumab is produced in the cell culture production medium at a titer of at least 10 g / L.

[0278] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium comprises a recombinant growth factor, an osmotic weight molar concentration regulator, a pH buffer, glutamine, methotrexate, and a cell protectant.

[0279] In one embodiment, the CHO cells are cultured at a temperature in the range of about 32°C to about 38°C.

[0280] In one aspect, a fed-batch production method for preparing dupilumab comprises culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding the dupilumab on a large scale in a cell culture production medium, wherein the sodium phosphate in the medium is supplemented to a concentration of about 200 to 550, about 200 to 275, about 250 to 325, about 300 to 375, about 350 to 425, about 400 to 475, about 450 to 525, or about 500 to 550 mg / L on days 2 and 4, or on days 0, 2, 4, 6, and / or 8, such that the titer of the dupilumab in the cell culture production medium on day 10 is about 5 g / L, about 6 g / L, about 7 g / L, or about 8 g / L. In another aspect, the titer of dupilumab in the cell culture production medium between day 10 and day 14 is about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, or about 10 g / L.

[0281] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium comprises a recombinant growth factor, an osmotic weight molar concentration regulator, a pH buffer, glutamine, methotrexate, and a cell protectant.

[0282] In one embodiment, the CHO cells are cultured at a temperature in the range of about 32°C to about 38°C.

[0283] In one embodiment, the sodium phosphate in the culture medium is supplemented at a concentration of about 200 to 550, about 200 to 275, about 250 to 325, about 300 to 375, about 350 to 425, about 400 to 475, or about 450 to 525 mg / L on days 2 and 4, or on days 0, 2, 4, 6, and / or 8.

[0284] In one aspect, a fed-batch production method for preparing dupilumab comprises culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding the dupilumab on a large scale in a cell culture production medium, wherein sodium phosphate in the medium is supplemented to a concentration of about 200 to 550, about 200 to 275, about 250 to 325, about 300 to 375, about 350 to 425, about 400 to 475, about 450 to 525, or about 500 to 550 mg / L on day 2 and day 4, or on days 0, 2, 4, 6, and 8, respectively, wherein tyrosine in the medium is supplemented at a concentration of about 2 g / L on day 3, and wherein insulin in the medium is supplemented at a concentration of about 7.5 mg / L on days 2 and 4, such that the titer of the dupilumab in the cell culture production medium on day 10 is at least 5 g / L.

[0285] In one aspect, a fed-batch production method for preparing dupilumab comprises culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding the dupilumab on a large scale in a cell culture production medium, wherein sodium phosphate in the medium is supplemented to a concentration of about 200 to 550, about 200 to 275, about 250 to 325, about 300 to 375, about 350 to 425, about 400 to 475, about 450 to 525, or about 500 to 550 mg / L on day 2 and day 4, or on days 0, 2, 4, 6, and 8, respectively, wherein tyrosine in the medium is supplemented at a concentration of about 1 g / L on day 3 and day 7, and wherein insulin in the medium is supplemented at a concentration of about 7.5 mg / L on day 2 and day 4, such that the titer of the dupilumab in the cell culture production medium on day 10 is at least 5 g / L.

[0286] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium comprises a recombinant growth factor, an osmotic weight molar concentration regulator, a pH buffer, glutamine, methotrexate, and a cell protectant.

[0287] In one embodiment, the CHO cells are cultured at a temperature in the range of about 32°C to about 38°C.

[0288] In one embodiment, the sodium phosphate in the culture medium is supplemented to a concentration of 200 to 550, about 200 to 275, about 250 to 325, about 300 to 375, about 350 to 425, about 400 to 475, or about 450 to 525 mg / L on days 2 and 4, or on days 0, 2, 4, 6, and 8.

[0289] In one embodiment, the culturing continues for about 10 days.

[0290] Various embodiments of the culture medium of the present invention include any combination of the above embodiments, including a chemically defined hydrolysate-free, serum-free culture medium comprising a specified amount of ornithine or putrescine and, in particular: (a) amino acids; (b) optional nucleosides; (c) salts of divalent cations; (d) fatty acids and tocopherols; and (e) vitamins. In some embodiments, a small amount of hydrolysate can be added to the PS culture medium.

[0291] Applicants contemplate that in the practice of the present invention, any one or more or combinations of a variety of basal media may be used to which ornithine or a combination of ornithine and putrescine is added. Basal media are generally known in the art and include Eagle MEME (minimum essential medium) (Eagle, Science, 1955, 112(3168):501-504), Ham F12 (Ham, Proc. Nat'l. Acad. Sci. USA, 1965, 53:288-293), F-12K medium, Dulbecco's medium, Dulbecco's modified Eagle's medium (Proc. Nat'l. Acad. Sci. USA, 1952, August; 38(8):747-752), 1:1 DMEM / Ham F12, Trowell T8, A2 medium (Holmes and Wolf, Biophys. Biochem. Cytol., 1961, 10: 389-401), Waymouth medium (Davidson and Waymouth, Journal of Biochemistry (Biochem. J.), 1945, 39 (2): 188-199), Williams E medium (Williams et al., Experimental Cell Research (Exp. Cell Res.), 1971, 69: 105 and below), RPMI1640 (Moore et al., Journal of the American Medical Association (J. Amer. Med. Assoc.), 1967, 199: 519524), MCDB 104 / 110 medium (Bettger et al., Proceedings of the National Academy of Sciences of the United States of America, 1981, 78 (9): 5588-5592), Ventrex HL-1 medium, albumin-globulin medium (Orr et al., Appl. Microbiol.), 1973, 25(1):4954), RPMI-1640 medium, RPMI-1641 medium, Iscove's Modified Dulbecco's Medium, McCoy 5A medium, Leibovitz L-15 medium, and serum-free medium, such as EX-CELLTM 300 series (JRH Biosciences, Lenexa, Kansas), protamine-zinc-insulin medium (Weiss et al., 1974, US 4,072,565), biotin-folic acid medium (Cartaya, 1978, US Re30,985), transferrin-fatty acid medium (Baker, 1982, US 4,560,655), transferrin-EGF medium (Hasegawa, 1982, US 4,615,977; Chessebeuf, 1984, US 4,786,599) and other media substitutes (see Inlow, US 6,048,728; Drapeau, US 7,294,484; Mather, US 5,122,469; Furukawa, US 5,976,833; Chen, US 6,180,401; Chen, US 5,856,179; Etcheverry, US 5,705,364; Etcheverry, US 7,666,416; Ryll, US 6,528,286; Singh, US 6,924,124; Luan, US 7,429,491, etc.).

[0292] In a specific embodiment, the culture medium is chemically defined and comprises, in addition to ornithine or a combination of ornithine and putrescine: CaCl 2 2H 2 O; HEPES buffer, KCl; MgSO 4 ;NaCl;Na 2 HPO 4 or other phosphates; pyruvate; L-alanine; L-arginine HCl; L-asparagine H 2 O; L-Aspartic acid; L-Cysteine ​​HCl H 2 O; L-glutamic acid; glycine; L-histidine HCl H 2 O; L-isoleucine; L-leucine; L-lysine HCl; L-methionine; L-ornithine HCl; L-phenylalanine; L-proline; L-serine; L-threonine; L-tryptophan; L-tyrosine 2Na2H 2O; L-valine; D-biotin; choline chloride; folic acid; inositol; niacinamide; pyridoxine HCl; D-pantothenic acid; riboflavin; thiamine HCl; vitamin B12; p-aminobenzoic acid; ethanolamine HCl; poloxamer 188; DL-a-tocopherol phosphate; linoleic acid; Na 2 SeO 3 ; lipoic acid; and glucose; and optionally adenosine; guanosine; cytidine; uridine; thymidine; and hypoxanthine 2Na.

[0293] In one embodiment, the starting osmolality of the culture medium is 200 to 500, 250 to 400, 275 to 350, or about 300 mOsm. During growth of the cells in the culture medium, and particularly after any feed according to a fed-batch regime, the osmolality of the culture may increase to about 350, 400, 450, or up to 500 mOsm.

[0294] In some embodiments where the osmolality of the defined medium is less than about 300, the osmolality is brought to about 300 by adding one or more salts in excess of a specified amount. In one embodiment, the osmolality is increased to a desired level by adding one or more osmotic agents selected from the group consisting of sodium chloride, potassium chloride, magnesium salts, calcium salts, amino acid salts, fatty acid salts, sodium bicarbonate, sodium carbonate, potassium carbonate, chelating agents as salts, sugars (e.g., galactose, glucose, sucrose, fructose, fucose, etc.), and combinations thereof. In one embodiment, the osmotic agent is added in excess and above its concentration in the components already present in the defined medium (e.g., sugars are added in excess and above the concentration specified for the sugar component).

[0295] The present disclosure provides a cell culture comprising a cell line expressing a protein of interest in an OS culture medium as described above. In one embodiment, the cell culture comprises insulin, which may be added to the culture medium as a point-of-use ingredient, or may be included in a culture medium formulation. In one embodiment, the cell line comprises cells capable of producing a biotherapeutic protein. Examples of cell lines routinely used to produce protein biotherapeutics include primary cells, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, 3T3 cells, HEK293 cells, RK cells, Per.C6 cells, and chicken embryo cells. In one embodiment, the cell line is a CHO cell line, or one or more of several specific CHO cell variants optimized for large-scale protein production, such as CHO-K1.

[0296] Animal cells (such as CHO cells) can be cultured in small-scale cultures, such as in a 125mL container with about 25mL culture medium, a 250mL container with about 50 to 100mL culture medium, or a 500mL container with about 100 to 200mL culture medium. Alternatively, the culture can be large-scale, such as a 1000mL container with about 300 to 1000mL culture medium, a 3000mL container with about 500mL to 3000mL culture medium, an 8000mL container with about 2000mL to 8000mL culture medium, and a 15000mL container with about 4000mL to 15000mL culture medium. The culture for manufacturing can include a large-scale 10,000L culture medium or more. Large-scale cell cultures such as those used for clinical manufacturing of protein therapeutics are usually maintained for several days or even weeks, while the cells produce the desired protein. During this time, the culture can be supplemented with a concentrated feed medium containing components consumed during the culture process, such as nutrients and amino acids. The concentrated feed medium can be based on any cell culture medium formulation. Such concentrated feed medium can, for example, contain most of the components of the cell culture medium in about 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 12 times, 14 times, 16 times, 20 times, 30 times, 50 times, 100 times, 200 times, 400 times, 600 times, 800 times or even about 1000 times its normal applicable amount. Concentrated feed medium is typically used in batch feed culture processes.

[0297] In some embodiments, during the process of cell growth or protein production, the cell culture medium is supplemented with "point-of-use additives", also referred to as additives, point-of-use ingredients, or point-of-use chemicals. Point-of-use additives include any one or more of the following: growth factors or other proteins, buffers, energy sources, salts, amino acids, metals, and chelators. Other proteins include transferrin and albumin. Growth factors, including cytokines and chemokines, are generally known in the art and are known to stimulate cell growth, or in some cases cell differentiation. Growth factors are typically proteins (e.g., insulin), small peptides, or steroid hormones, such as estrogen, DHEA, testosterone, and the like. In some cases, growth factors may be non-natural chemicals that promote cell proliferation or protein production, such as tetrahydrofolate (THF), methotrexate, and the like. Non-limiting examples of protein and peptide growth factors include angiogenin, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor (IGF), and glial cell line-derived neurotrophic factor (GDNF). F), migration stimulating factor, myostatin (GDF-8), nerve growth factor (NGF) and other neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), Wnt signaling pathway agonists, placental growth factor (PIGF), fetal bovine serum growth hormone (FBS), interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, etc. In one embodiment, the cell culture medium is supplemented with a point-of-use addition growth factor insulin. In one embodiment, the concentration of insulin in the culture medium, for example, the amount of insulin in the cell culture medium after addition, is about 0.1 μM to 10 μM. One or more of the point-of-use additions may also be included in the culture medium formulation of some embodiments.

[0298] Buffers are generally known in the art. The present invention is not limited to any one or any particular buffer, and any person of ordinary skill in the art can select an appropriate buffer or buffer system for use with a particular cell line producing a particular protein. In one embodiment, the point-of-use additive buffer is NaHCO 3 In one embodiment, the point-of-use addition buffer comprises NaHCO3 In another embodiment, the buffer is HEPES.

[0299] Energy sources suitable for use as point-of-use additives in cell culture are also well known in the art. Without limitation, in one embodiment, the point-of-use additive energy source is glucose. In view of the special and specific requirements of a particular cell line and protein to be produced, in one embodiment, glucose can be added to the culture medium at a cumulative concentration of about 1 to 35 mM. In some cases, glucose can be added at high levels of up to 10 g / L.

[0300] Chelating agents are also well known in the art of cell culture and protein production. Tetrasodium EDTA dihydrate and citrate are two common chelating agents used in the art, but other chelating agents can be used in the practice of the present invention. In one embodiment, the point-of-use additive chelating agent is tetrasodium EDTA dihydrate. In one embodiment, the point-of-use additive chelating agent is citrate, such as Na 3 C 6 H 5 O 7 .

[0301] In one embodiment, the cell culture may be supplemented with one or more point-of-use amino acids, such as glutamine. In one embodiment, the cell culture medium is supplemented with point-of-use addition glutamine at a final concentration of about 1 mM to 13 mM.

[0302] Other point-of-use additives include one or more of various amino acids and metal salts, such as salts of iron, nickel, zinc, and copper. In one embodiment, the cell culture medium is supplemented with any one or more of copper sulfate, zinc sulfate, ferric chloride, and nickel sulfate.

[0303] In one embodiment, the cell culture medium is supplemented with any one or more or all of the following point-of-use supplements: about 25 to 30 mM NaHCO3, about 1.5 to 2 mM glutamine, about 0.75 to 0.9 μM insulin, about 10.75 to 11.25 mM glucose, about 6.25 to 6.75 μM zinc sulfate, about 0.15 to 0.17 μM copper sulfate, about 70 to 80 μM ferric chloride, about 0.6 to 0.7 μM nickel sulfate, about 80 to 90 μM EDTA, and about 45 to 60 μM citrate.

[0304] In one embodiment, the culture medium is supplemented at certain time intervals during the cell culture according to a fed-batch process. Fed-batch culture is generally known in the art and is used to optimize protein production (see YM Huang et al., Biotechnol Prog. 2010 September to October; 26(5): 1400-10).

[0305] Cell viability, viable cell density, and cell doublings are improved relative to cells grown in medium without ornithine or putrescine. With respect to cell viability, cells grown in PS medium exhibit a viability that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, or at least 3 times greater than the viability of similar or identical cells grown in non-PS medium.

[0306] In some embodiments, the doubling rate of live mammalian cells in PS medium is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or at least 3 times greater than the doubling rate of mammalian cells cultured in non-PS medium. In some embodiments, the doubling rate of live mammalian cells in PS medium is about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% higher than the doubling rate of mammalian cells in non-PS medium.

[0307] In some embodiments, the doubling time of actively circulating mammalian cells in PS medium is less than 30 hours, less than 29 hours, less than 28 hours, less than 27 hours, less than 26 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, or less than 18 hours. In some embodiments, the doubling time of actively growing mammalian cells in PS medium is less than 28 hours. In some embodiments, the doubling time of mammalian cells in PS medium is about 27±1 hours, about 26±1 hours, about 25±1 hours, about 24±1 hours, about 23±1 hours, about 22±1 hours, or about 21±1 hours. In some embodiments, the doubling time of actively circulating mammalian cells in PS medium is about 24±1 hours. In some embodiments, the doubling time of actively dividing cells cultured in PS medium is at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, or at least 25% shorter than the doubling time of actively cycling cells cultured in non-PS medium.

[0308] Protein production

[0309] In addition to chemically defined PS medium and methods of culturing cells in PS medium, the present disclosure also provides methods of producing proteins, such as therapeutically effective antibodies or other biopharmaceutical drug substances, in cells cultured in PS medium.

[0310] In some embodiments, the protein production rate of mammalian cells cultured in PS medium is at least 5%, 10%, 15%, or 20% higher than the protein production rate of the same mammalian cells cultured in non-PS medium. In some embodiments, the protein production rate in cells cultured in PS medium is at least 1 pg / cell / day ("PCD"), at least 2 PCD, at least 3 PCD, at least 4 PCD, at least 5 PCD, at least 6 PCD, at least 7 PCD, at least 8 PCD, at least 9 PCD, at least 10 PCD, at least 15 PCD, at least 20 PCD, at least 25 PCD, at least 30 PCD, at least 35 PCD, at least 40 PCD, at least 45 PCD, at least 50 PCD, at least 75 PCD, or at least 100 PCD.

[0311] In some embodiments, the protein production yield or titer (which can be expressed as grams of protein product per liter of culture medium) from cells cultured in PS medium is at least 100 mg / L, at least 1 g / L, at least 1.2 g / L, at least 1.4 g / L, at least 1.6 g / L, at least 1.8 g / L, at least 2 g / L, at least 2.5 g / L, at least 3 g / L, at least 3.5 g / L, at least 4 g / L, at least 4.5 g / L, at least 5 g / L, at least 5.5 g / L, at least 6 g / L, at least 6.5 g / L, at least 7 g / L, at least 7.5 g / L, at least 8 g / L, at least 8.5 g / L, at least 9 g / L, at least 9.5 g / L, at least 10 g / L, or at least 20 g / L.

[0312] In some embodiments, the protein product (protein of interest) is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, an antigen-binding antibody fragment, a single-chain antibody, a bifunctional antibody, a trifunctional antibody or a tetrafunctional antibody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody.

[0313] In some embodiments, the protein of interest is a recombinant protein comprising an Fc portion and another domain. In some embodiments, the Fc portion comprises a hinge region followed by the CH2 and CH3 domains of IgG.

[0314] The present invention is not limited to any specific type of cells for protein production. Examples of cell types suitable for protein production include mammalian cells, primate cells, insect cells, avian cells, bacterial cells and yeast cells. The cell can be a stem cell or recombinant cell transformed by a vector for recombinant gene expression, or a cell transfected by a virus for producing a viral product. The cell can contain a recombinant heterologous polynucleotide construct encoding a protein of interest. The construct can be an episome, or it can be an element physically integrated into the genome of the cell. The cell can also produce a protein of interest without encoding the protein on a heterologous polypeptide construct. In other words, the cell can naturally encode a protein of interest, such as a B cell producing an antibody. The cell can also be a primary cell such as a chicken embryonic cell, or a primary cell line. Examples of suitable cells include BSC cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK-21 cells, chicken embryo cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, HEK293 cells, RK cells, Per.C6 cells and CHO cells. In various embodiments, the cell line is a CHO cell derivative, such as CHO-K1, CHO DUXB-11, Veggie-CHO, GS-CHO, S-CHO or CHO lec mutants.

[0315] In one embodiment, the cell is a CHO cell, which ectopically expresses the protein. In one embodiment, the protein comprises an immunoglobulin heavy chain region, such as a CH1, CH2 or CH3 region. In one embodiment, the protein comprises human or rodent immunoglobulin CH2 and CH3 regions. In one embodiment, the protein comprises human or rodent immunoglobulin CH1, CH2 and CH3 regions. In one embodiment, the protein comprises a hinge region and CH1, CH2 and CH3 regions. In some embodiments, the protein comprises an immunoglobulin heavy chain variable domain. In some embodiments, the protein comprises an immunoglobulin light chain variable domain. In some embodiments, the protein comprises an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain. In some embodiments, the protein is an antibody, such as a human antibody, a rodent antibody or a chimeric human / rodent antibody (e.g., human / mouse, human / rat or human hamster).

[0316] The production phase can be carried out at any culture scale, from flasks or wave bags, to one liter bioreactors, to large-scale industrial bioreactors. Large-scale processes can be carried out in volumes of about 1,000 liters to 25,000 liters or more. One or more of several means can be used to control protein production, such as temperature changes or chemical induction. The growth phase can occur at a higher temperature than the production phase. For example, the growth phase can occur at a first temperature of about 35°C to 38°C, and the production phase can occur at a second temperature of about 29°C to 37°C, optionally at about 30°C to 36°C or about 30°C to 34°C. In addition, chemical inducers of protein production, such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline and hexamethylenebisacetamide (HMBA) can be added while, before or after the temperature change. If an inducer is added after the temperature change, it can be added one hour to five days after the temperature change, such as one to two days after the temperature change. Production cell cultures can be operated as a continuous feed culture system, such as in a chemostat (see C. Altamirano et al., Biotechnol Adv. 2001 Nov-Dec;17(6):1032-41), or according to a fed-batch process (Huang, 2010).

[0317] The present invention is applicable to improving protein production via cell culture processes. The cell lines used in the present invention can be genetically engineered to express polypeptides of commercial or scientific interest. Genetically engineered cell lines involve transfection, transformation or transduction of cells with recombinant polynucleotide molecules, or otherwise alteration (e.g., by homologous recombination and gene activation or fusion of recombinant cells with non-recombinant cells) to allow host cells to express the desired recombinant polypeptide. Methods and vectors for genetically engineering cells or cell lines to express polypeptides of interest are well known to those skilled in the art; for example, various technical descriptions are in the following documents: Molecular Biology Laboratory Guide (Current Protocols in Molecular Biology.) Ausubel et al., ed., (Wiley & Sons, New York, 1988 and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15-69. A wide variety of cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, VA) and commercial suppliers. Examples of cell lines commonly used in industry include VERO, BHK, HeLa, CVI (including Cos), MDCK, HEK293, 3T3, myeloma cell lines (e.g., NSO, NSI), PC12, W138 cells, and Chinese hamster ovary (CHO) cells. CHO cells are widely used to produce complex recombinant proteins, such as cytokines, coagulation factors, and antibodies (Brasel et al. (1996), Blood 88:2004-2012; Kaufman et al. (1988), J. Biol Chem 263:6352-6362; McKinnon et al. (1991), J Mot Endocrinol 6:231-239; Wood et al. (1990), J Immunol. 145:3011-3016). A mutant cell line deficient in dihydrofolate reductase (DHFR) (Urlaub et al. (1980), Proc. Natl. Acad. Sci. USA 77:4216-4220) DXBI 1 is a desirable CHO host cell line because an efficient DHFR selectable and amplifiable gene expression system allows high-level recombinant protein expression in these cells (Kaufman RJ. (1990), Meth Enzymol 185:537-566).In addition, these cells are easy to manipulate as attached or suspended cultures and present relatively good gene stability. CHO cells and their recombinantly expressed proteins have been extensively characterized and approved for commercial manufacturing. In some embodiments, the CHO cell line is a cell line described in U.S. Patent Application Publication Nos. 2010 / 0304436A1, 2009 / 0162901A1 and 2009 / 0137416A1 and U.S. Patent Nos. 7,455,988B2, 7,435,553B2 and 7,105,348B2.

[0318] The present disclosure is not limited to the scope of the specific embodiments described herein, which are intended as illustrations of individual aspects or embodiments of the present invention. Functionally equivalent methods and components are within the scope of the present invention. In addition to the modifications described herein, various modifications of the present invention will be apparent to those skilled in the art based on the foregoing description and the accompanying drawings. Such modifications are within the scope of the present invention.

[0319] The present disclosure is based in part on the discovery that the addition of ornithine or a combination of ornithine and putrescine to serum-free cell culture media results in increased cell growth, survival, and polypeptide production from recombinantly engineered animal cell lines (or natural cells) expressing a protein of interest, thereby enhancing culture stability and increasing the yield of the polypeptide of interest.

[0320] Seed expansion culture optimization and measurement

[0321] As described herein, an improved method for optimizing the large-scale production of protein in cell culture by implementing a seed-enlarged culture process has been developed, and the seed-enlarged culture process forces cells and biases cells to increase the viable cell density (VCD) in a large-scale production container. In the U.S. application No. 16 / 984,581 (which is incorporated by reference) of Metabolically Optimized Cell Culture, it has been previously observed that the peak lactic acid consumption in the seed-enlarged culture can be used to improve the large-scale production of protein. As described herein, a new seed-enlarged culture process has been developed, which further enhances cell metabolism and productivity by adjusting the initial VCD and without affecting the observed lower peak lactic acid consumption, which confirms a novel and different mechanism for enhancing protein production compared to the mechanism described in U.S. application No. 16 / 984,581.

[0322] By way of background technology, it is well known that protein production via cell culture is usually carried out using a batch or batch feed process. The early stages of inoculum growth after vial thawing include culturing cells in seed expansion culture. Culture vessels include, but are not limited to, well plates, wave bags, T bottles, shake flasks, stirred vessels, spinner bottles, hollow fibers, airlift bioreactors, etc. Suitable cell culture vessels are bioreactors. A bioreactor refers to any culture vessel that is manufactured or engineered to manipulate or control environmental conditions. Such culture vessels are well known in the art. Typically, cells grow at an exponential growth rate such as in a seed expansion culture bioreactor so as to gradually increase the size and / or volume of the cell population. After the cell mass is scaled up via several bioreactor stages, the cells are transferred to a production bioreactor while the cells are still in exponential growth (logarithmic phase) (Gambhir, A. et al., 2003, Journal of Bioscience and Bioengineering (J Bioscience Bioeng) 95 (4): 317-327). It is generally considered undesirable for cells in batch cultures (e.g., seed cultures) to pass beyond the logarithmic phase into the stationary phase. It has been suggested that cultures should be passaged while they are in the logarithmic phase, before cells (e.g., adherent cells) reach confluence, because contact inhibition or waste accumulation inhibits cell growth, among other reasons (Cell Culture Basics, Gibco / Invitrogen Online Handbook, www.invitrogen.com; Animal Cell Culture Guide, www.atcc.org).

[0323] After transfer to fed-batch culture, the cells are cultured for a period of time while monitoring and controlling the composition of the culture medium to allow production of the protein or polypeptide of interest. After a specific yield is reached, or cell viability, waste accumulation, or nutrient consumption determine that the culture should be terminated, the produced protein or polypeptide is isolated. Efforts to reduce the output of metabolic waste products (such as lactic acid accumulation) in cell culture, in particular, have improved overall final protein titer. These efforts have focused on controlling glucose- or nutrient-limited batch feeding processes (see, e.g., WO2004104186; U.S. Pat. No. 8,192,951 B2), improving cell culture conditions (e.g., U.S. Pat. No. 7,390,660; Zagari et al., 2013, New Biotechnol., 30(2):238-45), or cell engineering, including targeting enzymes in the glycolytic pathway (e.g., Kim, SH and Lee, GM, 2007, Appl. Microbiol. Biotechnol. 74, 152-159; Kim, SH and Lee, GM, 2007, Appl. Microbiol. Biotechnol. 76, 659-665; Wlaschin, KF and Hu, WS., 2007, J. Biotechnol. 131, 168-176).

[0324] Controlling the feeding of cells is used to target a more efficient metabolic phenotype (Europa, AF et al., 2000, Biotechnol. Bioeng. 67:25-34; Cruz et al., 1999, Biotechnol. Bioeng. 66(2):104-113; Zhou et al., 1997, Cytotechnology 24, 99-108; Xie and Wang, 1994, Biotechnol. Bioeng. 43:1174-89). However, this control is complicated by the fact that nutrient deprivation and rapid changes in ammonia concentration, such as those seen in high cell density fed-batch cultures, can induce apoptosis ("programmed cell death") (Newland et al., 1994, Biotechnol. Bioeng. 43(5):434-8). Therefore, a common optimization process is to grow cells to a suitably high density in a fed-batch system and then intentionally induce a prolonged production plateau by, for example, temperature or pH shifts (Quek et al., 2010, Metab Eng 12(2):161-71. DOI: 10.1016 / j.ymben.2009.09.002. Epub 2009 Oct 13).

[0325] In some embodiments, the initial VCD in the seed expansion process is adjusted to enhance protein production in a batch or fed-batch process. In some aspects, the initial VCD at each step (from N-5 to N-1) ranges from 1.7×10 5 cells / mL to 2.3×10 5 The initial VCD increased to approximately 3.5 × 10 cells / mL compared to the acceptable standard initial VCD of 5 cells / mL to 5.43×10 5 In some aspects, the initial VCD is about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 times higher than an alternative initial VCD in an acceptable standard seed expansion culture.

[0326] In some embodiments, compared with standard seed expansion culture, the initial VCD that increases N-5 to N-1 container does not produce change to the final VCD of N-1. In one aspect, compared with standard seed expansion culture, the seed expansion culture of optimization causes the final VCD in N-5 to N-2 container to increase. In one aspect, the peak lactic acid observed in the final production container does not have substantial difference compared with standard seed expansion culture. In one aspect, the continuous biomass of optimization seed expansion culture increases. In one aspect, the seed expansion culture of optimization causes final titer (g / L) to increase by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0327] Example 22 is provided to describe to one of ordinary skill in the art how to scale up cultures using optimized seeds, and is not intended to limit the scope of what the inventors regard as their invention.

[0328] Due to the importance of VCD in seed expansion culture process and in-process control, a more efficient and improved method for measuring VCD is needed. Currently, the determination for monitoring the VCD of suspension mammalian cell line cell culture is based on offline analysis (Ritacco, Biotechnology Progress, 34 (6): 1407-1426, 2018). For example, the trypan blue exclusion test (TBET) can be visually distinguished between live cells and non-living cells using a manual hemocytometer or an automated bioanalyzer; however, offline determination does not comply with the U.S. Food and Drug Administration (Food and Drug Administration; FDA) regulatory framework, which is implemented via online or on-line measurement of key process parameters (CPPs) affecting key quality attributes (CQAs) to design, analyze and control mechanisms for pharmaceutical manufacturing processes (e.g., process analytical technology / (PAT)) (Metze, Bioprocess and Biosystems Engineering, 43 (2): 193-205, 2020). Offline assays also introduce a delay between sampling and reacting to the process, and require more labor and resources.

[0329] It will be appreciated that on-line and / or at-line methods and systems for measuring VCD are needed to optimize cell culture therapeutic protein production and adhere to the regulatory framework outlined by the FDA for the implementation of process analytical techniques.

[0330] Dielectric spectroscopy is a promising technology for real-time analysis of cell culture and biomass properties. Dielectric spectroscopy is a non-destructive, non-invasive continuous process monitoring tool that can generate continuous process data for both suspension and attached cell cultures. However, the implementation of biomass on-line monitoring technology is still challenging due to the complexity of the relevant calibration process, the difficulty of integration and the lack of model accuracy and transferability (Carvell, Cell Technology, 50: 35-48, 2006, Kell et al., Journal of Bioelectricity (J. Bioelectricity), 4 (2): 317-348, 1990). Therefore, the present application provides the benefits of dielectric spectroscopy systems and methods that can accurately determine on-line live cell density predictions during cell culture.

[0331] In one embodiment, the present disclosure provides a method for culturing cells by: measuring a first property of the cell culture; using the first property to predict a second property of the cell culture; and adjusting culturing conditions based on the predicted second property of the cell culture.

[0332] In one embodiment, the capacitive probe may be a capacitive or dielectric sensor that generates an alternating dielectric field and measures the permittivity of the surrounding medium. The capacitive probe may use at least one electrode to generate an alternating electric field. The electrodes within the capacitive probe may include platinum, aluminum, gold, or other suitable metals.

[0333] In one aspect, an inline sensor is used to measure a first property of a cell culture.

[0334] In one aspect, a correlation equation is used to predict a second property of the cell culture based on a measured first property of the cell culture.

[0335] In one aspect, the cell culture property can be capacitance, viable cell density, permittivity, and / or conductivity.

[0336] In one embodiment, the present disclosure provides a method for measuring VCD of cells cultured in a bioreactor by applying an electric field to cells cultured in the bioreactor, measuring capacitance, and correlating the measured capacitance with viable cell density.

[0337] In one embodiment, the present disclosure provides a method for culturing cells by: measuring a first characteristic of the cell culture using an inline sensor; predicting at least one measurement of a second characteristic of the cell culture using the measurement of the first characteristic of the cell culture and a correlation equation; and adjusting culture conditions used to culture the cells based on the at least one predicted measurement of the second characteristic of the cell culture.

[0338] In one aspect, an offline assay is used to measure a second property of the first cell culture.

[0339] In one aspect, the cells are from the same cell line as that used to derive the correlation equation.

[0340] In one aspect, the cells are from a different cell line than the cell line used to derive the correlation equation.

[0341] In one aspect, the correlation equation is derived using more than one cell line.

[0342] In one aspect, more than one first capacitance value is measured for the first cell culture.

[0343] In one aspect, more than one first VCD value is measured for the first cell culture.

[0344] In one aspect, at least 50% of the variability in the first VCD value is attributable to changes in the first capacitance value.

[0345] In one aspect, the correlation equation is generated using multivariate data analysis.

[0346] In one embodiment, a bioanalyzer system can be used to perform bioanalyzer measurements of cell culture samples. The bioanalyzer system can include at least one 96-well plate, a syringe, a 24-position external sample tray, a lab-on-a-chip, or a combination thereof. The bioanalyzer system for performing bioanalyzer measurements of cell culture samples can be automated. The bioanalyzer system can analyze a single sample by performing about 1 to about 20 assays simultaneously. Exemplary non-limiting bioanalyzer measurements that can be performed by the bioanalyzer system include measurements of pH, CO 2 The partial pressure (pCO 2 ), O 2 The partial pressure of glucose, lactate, glutamine, glutamate, ammonium, sodium, potassium, calcium, osmolality, total cell density, viable cell density, cell viability, or a combination thereof can be quantified. The bioanalyzer system can use a trypan blue exclusion test to determine the viable cell density. The cell culture sample in the on-chip laboratory can be analyzed using at least one electrophoretic separation, flow cytometric analysis technology, or a combination thereof. Electrophoretic separation can be used to perform DNA, RNA, or protein determinations, or a combination thereof. DNA determinations can determine the size or quantity of DNA, or the size and quantity of DNA in a cell culture sample. RNA determinations can quantify total RNA, mRNA, or RNA and mRNA in a sample, determine the size of at least one small RNA in a sample, determine the purity and integrity of a sample, or a combination thereof. Protein determinations can determine the size or quantity of at least one protein in a sample. Flow cytometric analysis techniques can be used to analyze protein expression, cell apoptosis, gene silencing transfection monitoring, intracellular staining, extracellular staining, or a combination thereof.

[0347] In one aspect, the cell culture nutrients may include, for example, glucose, glutamine, glutamate, sodium, potassium, calcium, O 2 or a combination thereof.

[0348] In one aspect, cell culture metabolites may include, for example, acetate, acetate, lactate, lactate, ammonia, ethanol, lactate, CO 2 or a combination thereof.

[0349] In one aspect, viable cell density refers to the number of viable cells in a given volume of culture medium, as determined by measuring the permittivity of the surrounding medium, standard viability assays (including trypan blue dye exclusion tests), or a combination thereof.Cell density refers to the number of cells in a given volume of culture medium.

[0350] Example 23 is provided to describe to one of ordinary skill in the art how to use an improved capacitance probe to measure VCD, and is not intended to limit the scope of what the inventors regard as their invention.

[0351] Optimization of vessel and bioreactor stirring, sparging and measurements

[0352] It is important to optimize the starting volume, air sparging, and agitation set points in the containers and bioreactors because during the growth and production phases, the cell culture is exposed to shear stress from the agitator impeller and sparging gas, which is required to maintain adequate oxygen delivery and stripping of dissolved carbon dioxide to maintain cell health. It has been found that these processes can be optimized by using stepped or different agitation and air sparging rates. For example, it has been found that stepped or different agitation and air sparging rates provide greater improvements in VCD. Dissolved oxygen and carbon dioxide can be controlled at set points via sparging on a cascade control loop.

[0353] It has also been found that by reducing the amount of acidic and basic species of the recombinant protein of interest, stepwise or altered stirring and air bubbling rates can reduce heterogeneity. These variants can result from deamidation, sialylation, glycosylation, and fragmentation, which can change the stability, activity, and efficacy of proteins containing the Fc portion (part of the crystallizable fragment region from the antibody). Sissolak et al., Journal of Industrial Microbiology and Biotechnology (J. Indust. Microbiol. Biotech.) 46: 1167-78 (2019). Basic variants can increase the binding of antibodies to Fc receptors. Hintersteiner et al., MABS 8: 1458-60 (2016).

[0354] Fc glycans can play a role in immunogenicity, bioactivity, pharmacodynamics, and pharmacokinetics. Reusch and Tejada, Glycobiol. 25: 1325-34 (2015). The phenomenon that may occur is called non-glycosylated heavy chain (NGHC). NGHC variation can change effector functions, such as opsonization. Opsonization involves the Fc part involved in ADCC (antibody-dependent cellular toxicity), ADCP (antibody-dependent cellular phagocytosis), and CDC (complement-dependent cytotoxicity). Therefore, depending on the mode of action, it may be necessary to control charge variation and NGHC in proteins containing the Fc part.

[0355] In an exemplary embodiment, the agitation of the culture is increased by 25%, 50%, 75%, 100%, 125%, 150%, 175% or 200% on day 0.5, day 1, day 1.5, day 2, day 2.5, day 3, day 3.5, day 4, day 4.5, day 5, day 5.5, day 6, day 6.5, day 7, day 7.5, day 8, day 8.5, day 9, day 9.5, day 10, day 10.5 or day 11. In one aspect, the initial agitation rate is between 20 rpm and 150 rpm, and is increased to 40 rpm to 300 rpm, and then increased again to 80 rpm to 600 rpm. The agitation rpm can also be related to power per unit volume using known models. For example, in a 10,000 L bioreactor with two impellers spinning at 40 rpm at 10,000 L, the power per unit volume equals 0.076 hp / 1000 L; when spinning at 22 rpm at 7,500 L, the power per unit volume equals 0.017 hp / 1000 L. In a 3,000 L bioreactor with two impellers spinning at 40 rpm at 3,000 L, the power per unit volume equals 0.033 hp / 1000 L; when spinning at 40 rpm at 2,500 L, the power per unit volume equals 0.04 hp / 1000 L. In a 10,000 L bioreactor with a single impeller spinning at 40 rpm at 10,000 L, the power per unit volume equals 0.005 hp / 1000 L; when spinning at 22 rpm at 7,500 L, the power per unit volume equals 0.001 hp / 1000 L.

[0356] In another aspect, the initial stirring rate is about 75 rpm and is then increased to 150 rpm and then increased to 225 rpm. In an exemplary embodiment, the air sparging rate of the culture is increased by 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% on day 0.5, day 1, day 1.5, day 2, day 2.5, day 3, day 3.5, day 4, day 4.5, day 5, day 5.5, day 6, day 6.5, day 7, day 7.5, day 8, day 8.5, day 9, day 9.5, day 10, day 10.5, or day 11. In one aspect, the initial bubbling rate is between 50 slpm and 75 slpm, and increases to about 225 slpm to 275 slpm on day 2. In one aspect, the bubbler has between 146 and 292 holes with a size between 0.5 mm and 2 mm, and may include, for example, bubblers with 146×0.5 mm holes, 146×1.0 mm holes, 146×1.5 mm holes, 146×2.0 mm holes, 292×0.5 mm holes, 292×1.0 mm holes, 292×1.5 mm holes, and 292×2.0 mm holes.

[0357] In one aspect, the stirring rate and the air sparging rate are increased on the same day. In another aspect, the stirring rate and the air sparging rate are increased on different days, wherein the stirring rate can be as follows: Fig.45 The results shown in Figure 2 show an increase on the feed day.

[0358] In one exemplary embodiment, the starting capacity of a vessel or bioreactor can be optimized to reduce stirring-related surface effects. The initial working capacity inside a vessel or bioreactor is typically set at a level with sufficient empty space to provide room for capacity adjustments, including subsequent feeding. It was unexpectedly found that increasing the initial working capacity increased biomass and final titer. It was found that by increasing the working capacity, it reduced stirring-related surface effects caused by the impeller in the bioreactor, including the air cascade effect, and provided better control of dissolved oxygen concentration.

[0359] In one aspect, the initial working capacity of the container or bioreactor is adjusted to ensure that the uppermost impeller (if more than one) is covered by cell culture medium at the start of the culturing process. Examples of different containers and optimized capacities (kg) to reduce stirring-related surface effects are shown in Table 6 below.

[0360] Table 6

[0361] Unit Operation Original capacity (kg) (range) Optimized capacity (kg) (range) 50L 40-45 45-50 500L 425-460 460-480 3,000L 1,775-1,825 1,850-1,900 10,000L 7,000-7,400 7,400-7,800

[0362] In one embodiment, the dissolved oxygen level is measured periodically. In one aspect, the period for measuring the dissolved oxygen level can be selected by the user. In another aspect, the dissolved oxygen level can be automatically measured at a set time period. In one aspect, the sparging rate can be configured to automatically adjust the sparging rate based on the measured dissolved oxygen level to maintain a set level of dissolved oxygen, which can be constant, variable, or stepped using cascade control as described above.

[0363] As described above, dissolved oxygen and carbon dioxide concentrations are key process parameters that must be carefully controlled, which also require optimized systems and methods for measuring dissolved gases. In one embodiment, an improved probe for measuring dissolved gases in large-scale containers and bioreactors for culturing cells in the manufacture of therapeutic protein products in the biopharmaceutical industry is disclosed. An improved data processing method is generated by comparing data from two optical probes (referred to as optical probe A and optical probe B) with electrochemical probe data in large-scale industrial bioreactors. As shown in Examples 19 and 21 below, a calibration method is generated to take into account the offset observed between the two optical probes relative to the electrochemical probe (optical probe A: ~ +1.6% saturation, optical probe B: ~ -3.0% saturation), to achieve the goal of obtaining the necessary accuracy while minimizing maintenance and reducing or eliminating unwanted offset events outside the normal operating range. It is worth noting that the optical probe exhibits 100% offset elimination above the normal operating range observed on the electrochemical probe, and approximately twice the noise reduction compared to the electrochemical probe (based on standard deviation comparison).

[0364] Additionally, as demonstrated in Example 20, an improved signal processing method is generated that includes filtering and smoothing the data to allow the electrochemical probe to reduce excursion events determined as false positives. In one aspect, the signal processing is applied in real time.

[0365] In one embodiment, cells are cultured in a large-scale container or bioreactor where the content of dissolved gases (including oxygen) is measured periodically. In one aspect, the period for measuring dissolved oxygen levels can be selected by the user. In another aspect, dissolved oxygen levels can be measured automatically at set time periods.

[0366] In one embodiment, a container or bioreactor with reduced maintenance requirements is configured to measure dissolved oxygen using one or more optical probes. Bioreactors and containers may also be configured to have one or more optical probes to measure other gases, such as carbon dioxide. In one aspect, data from the optical probe is processed based on a fixed offset. In one aspect, the offset is based on the correlation of the data from the optical probe and the electrochemical probe. In another aspect, the offset is based on the correlation with the optical probe and a known standard. In another aspect, data from the optical probe is processed based on an offset that varies over time during the culture process. In one aspect, the offset is applied in real time.

[0367] In another embodiment, the vessel or bioreactor is configured to use one or more electrochemical probes with optimized data processing by filtering and smoothing the signals from the electrochemical probes. In one aspect, the sampling window for smoothing the data is between a 10 minute window and a 33 minute window.

[0368] It is believed that the same methods used to improve the accuracy and reduce signal noise of optical and electrochemical probes used to measure dissolved oxygen can also be applied to measuring carbon dioxide and other dissolved gases.

[0369] In one embodiment, cells are grown at an appropriate pCO 2 The cells may be cultured under conditions that control the heterogeneity of antibodies and derivatives and fragments of both produced recombinantly in mammalian cells and are more fully described in U.S. Patent Application No. 63 / 246,047 (Methods of Controlling Antibody Heterogeneity), which is incorporated herein by reference. The cells may be any suitable mammalian cell, including CHO, BHK, HEK293, HeLa, human amnion, Per.C6, and Sp2 / 0 cells. While not being bound by any theory, it is believed that increasing CO in the culture medium 2 The content will cause intracellular CO 2 Increases in pH, which may cause charge and glycosylation variations. This effect is separate from any decrease in pH that may be caused by the formation of carbonic acid.

[0370] Carbon dioxide concentration can be measured using CO 2 Bubbling or by reducing air sparging. Reducing the pressure in the production bioreactor will reduce the solubility of oxygen; this in turn requires more sparging of more oxygen to maintain the dissolved oxygen (DO) set point and increased gas flow rate, which in turn reduces the pCO2 in the culture medium. Carbon dioxide concentration can be measured using a CO2 electrode (also known as a Severinghaus electrode). More advanced systems are commercially available, such as FLEX and FLEX 2 analyzers. Charge variants can be measured using Imaged Capillary Isoelectric Focusing (iCIEF) and ion exchange chromatography with salt gradient elution. NGHC can be measured by reducing capillary electrophoresis (CE)-SDS.

[0371] The cells may be cultured for about 10 to 15 days. In one embodiment, the cells may be cultured for about 14 days. During the culture period, the pCO2 conditions may be between about 30 mmHg and about 210 mmHg, between 50 mmHg and 200 mmHg, between 60 mmHg and 190 mmHg, between 70 mmHg and 180 mmHg, between 80 mmHg and 170 mmHg, between 90 mmHg and 160 mmHg, between 100 mmHg and 150 mmHg, between 110 mmHg and 140 mmHg, between 120 mmHg and 140 mmHg, between 120 mmHg and 130 mmHg, or any value within these ranges, preferably maintained by CO2 bubbling, and may be measured using a CO2 electrode.

[0372] The method may include inoculating a culture medium with mammalian cells that produce an Fc-containing protein, such as an antibody capable of binding to an IL-4 receptor; and culturing the cells under pCO2 conditions that allow the mammalian cells to produce an Fc-containing protein capable of binding to an IL-4 receptor. Preferably, the protein is a human monoclonal antibody, preferably an IgG antibody, including subclasses such as IgG1 and IgG4.

[0373] In another aspect, the main peak form of the Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, produced by the cell may comprise between about 38% and about 65% of the total Fc-containing protein; the acidic variant of the Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, may comprise between about 20% and about 47% of the total Fc-containing protein; and the basic variant of the Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, may comprise up to about 36% of the total Fc-containing protein. The percentage of Fc-containing protein with non-glycosylated heavy chains may comprise between about 3% and about 8%, 4% to 7%, 5% to 7% and 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or fractional value within these ranges.

[0374] In another aspect, the main peak form of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, produced by the cells may comprise between about 50% and about 70% of the total Fc-containing proteins, the acidic variants of the Fc-containing proteins may comprise between about 20% and about 47% of the total Fc-containing proteins, and the basic variants of the Fc-containing proteins may comprise up to about 15% of the total Fc-containing proteins, such as antibodies, total antibodies, antibody derivatives, or antibody fragments capable of binding to IL-4 receptor. The percentage of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, having non-glycosylated heavy chains may comprise between about 3% and about 8%, 4% to 7%, 5% to 7%, and 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or fractional value within these ranges.

[0375] In another aspect, the main peak form of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, produced by the cells may comprise between about 50% and about 70% of the total Fc-containing proteins, the acidic variants of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, may comprise between about 20% and about 47% of the total Fc-containing proteins, and the basic variants of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, may comprise up to about 6%, about 8%, or about 10% of the total Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, total antibodies, antibody derivatives, or antibody fragments. The percentage of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, having non-glycosylated heavy chains may comprise between about 3% and about 8%, 4% to 7%, 5% to 7%, and 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or fractional value within these ranges.

[0376] In another aspect, the main peak form of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, produced by the cells may comprise between about 50% and about 65% of the total Fc-containing proteins, such as antibodies, total antibodies, antibody derivatives, or antibody fragments capable of binding to IL-4 receptor, acidic variants of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, may comprise between about 23% and about 46% of the total Fc-containing proteins, and basic variants of Fc-containing proteins may comprise up to about 15% of the total Fc-containing proteins, such as antibodies, total antibodies, antibody derivatives, or antibody fragments capable of binding to IL-4 receptor. The percentage of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, having non-glycosylated heavy chains may comprise between about 3% and about 8%, 4% to 7%, 5% to 7%, and 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or fractional value within these ranges.

[0377] In another aspect, the main peak form of the Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, produced by the cell may comprise between about 50% and about 65% of the total Fc-containing protein, the acidic variant of the Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, may comprise between about 31% and about 46% of the total Fc-containing protein, and the basic variant of the Fc-containing protein may comprise up to about 15% of the total Fc-containing protein, such as an antibody, total antibody, antibody derivative or antibody fragment capable of binding to IL-4 receptor. The percentage of Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, having a non-glycosylated heavy chain may comprise between about 3% and about 8%, 4% to 7%, 5% to 7% and 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5% or any integer or fractional value within these ranges.

[0378] In another aspect, the main peak form of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, produced by the cells may comprise between about 50% and about 65% of the total Fc-containing proteins, the acidic variants of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, may comprise between about 23% and about 39% of the total Fc-containing proteins, and the basic variants of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, may comprise up to about 15% of the total Fc-containing proteins, such as antibodies, total antibodies, antibody derivatives or antibody fragments capable of binding to IL-4 receptor. The percentage of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, having non-glycosylated heavy chains may comprise between about 3% and about 8%, 4% to 7%, 5% to 7% and 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or fractional value within these ranges.

[0379] In another aspect, the main peak form of the Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, produced by the cell may comprise between about 35% and about 60% of the total Fc-containing protein, the acidic variant of the Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, may comprise between about 20% and about 40% of the total Fc-containing protein, and the basic variant of the Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, may comprise between about 10% and about 40% of the total Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, total antibody, antibody derivative or antibody fragment. The percentage of Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, having a non-glycosylated heavy chain may comprise between about 3% and about 8%, 4% to 7%, 5% to 7% and 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or fractional value within these ranges.

[0380] In another aspect, the main peak form of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, produced by the cells may comprise between about 39% and about 50% of the total Fc-containing proteins, the acidic variants of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, may comprise between about 22% and about 38% of the total Fc-containing proteins, and the basic variants of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, may comprise between about 14% and about 36% of the total Fc-containing proteins, such as antibodies, total antibodies, antibody derivatives or antibody fragments capable of binding to IL-4 receptor. The percentage of Fc-containing proteins, such as antibodies capable of binding to IL-4 receptor, having non-glycosylated heavy chains may comprise between about 3% and about 8%, 4% to 7%, 5% to 7% and 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or fractional value within these ranges.

[0381] Additional details based on FcR-containing proteins, such as antibodies capable of binding to the IL-4 receptor, are further described by Example 18 below, which illustrates various aspects of the invention but does not limit the invention in any way and can be applied to the appropriate pCO 2 Cells cultured under conditions used to control for acidic species and unglycosylated heavy chain of dupilumab.

[0382] In an exemplary embodiment, the glucose feeding strategy is also optimized to prevent high osmotic weight molar concentration conditions. It is found that glucose concentration affects osmotic conditions, which can affect cell culture performance. To improve cell culture conditions and performance, the lower dextrose target is reduced to prevent high osmotic weight molar concentration conditions. In one aspect, the dextrose target is reduced by 10%, 20%, 30%, 40%, 50%, 60% or 70%. In one aspect, the dextrose target is gradually increased on the 2nd day, the 3rd day, the 4th day, the 5th day or the 6th day. In one aspect, the dextrose target is gradually increased and then reduced after the 0.5th day, the 1st day, the 1.5th day, the 2nd day, the 2.5th day or the 3rd day. In an exemplary embodiment, the initial dextrose target is between 5g / L and 7g / L, and the dextrose target that is gradually increased is between 7g / L and 11g / L. In one embodiment, the dextrose target is gradually increased and gradually reduced once or multiple times. Fig.58A and Fig.58B Exemplary dextrose targets and regulation over time are shown in .

[0383] In another aspect, the seed expansion culture and production medium and feed can be subjected to a high temperature short time (HTST) treatment at 102° C. for 10 seconds. In another aspect, the HTST treatment is performed at a temperature of about 101° C., 102° C., 103° C., 104° C., 105° C., or about 106° C. for a time of 8 seconds, 9 seconds, 11 seconds, 12 seconds, 13 seconds, 14 seconds, or 15 seconds. HTST can serve as a rapid pasteurization of cell culture media and feeds, which can be used in manufacturing as a viral barrier for upstream processing and reduce adventitious factor risks, resulting in equivalent process performance and product quality, including titer.

[0384] Centrifugation, depth and fine filtration

[0385] In certain exemplary embodiments, a recovery may include one or more centrifugation steps, including disc-stack centrifugation and depth, polish and guard filtration or direct depth and polish filtration without centrifugation, to separate the protein of interest from host cells and accompanying cell debris. Fig.28 A schematic diagram showing an exemplary overview of the use of depth filters, fine filters and protection filters is shown in FIG.

[0386] Centrifugation of the sample can be performed at, for example, (but not limited to) 7,000×g to about 12,750×g. It is recommended to fill the drum with 60% to 80% solids in order to calculate the discharge interval. The centrate is then processed by online depth, fine and protection filtration. Therefore, the filter flux varies with the centrifuge feed flow rate and the area of ​​the depth, fine and protection filters. In some embodiments, a drum speed of 5550rpm (7000g) and a feed flow rate of about 1630 (L / h) can be used. The filter flux and discharge interval can be calculated as shown in Table 7 below:

[0387] Table 7

[0388]

[0389]

[0390] g, standard gravity

[0391] It has been found that the drum speed can affect turbidity by changing the sedimentation rate of particles of various sizes. In the case of large-scale production, such centrifugation can be set to be carried out on-line at a flow rate that achieves a turbidity level of, for example, 150 NTU in the resulting supernatant.

[0392] The cell culture fluid or centrate may need to be adjusted to a specific pH and conductivity in order to obtain the desired impurity removal and product recovery from the depth filtration step.

[0393] To reduce batch-to-batch variation, the centrate turbidity should be measured at the midpoint between discharges, referred to as the drum midpoint ((t=1 / 2 discharge interval)), to determine if adjustments to the drum rotation speed and feed flow rate need to be made. It has been found that faster flow rates result in shorter residence times, which can increase turbidity and reduce turbidity clearance relative to the filter. It has been found that because the amount of shear induced by centrifugation changes with changes in drum rotation speed and therefore changes the particle size distribution, centrifuge performance cannot be predicted by linear extrapolation using the delta concept alone.

[0394] To overcome these limitations, a predictive profiler was created to observe Fig.29 The factors and reactions reflected in it.

[0395] The obtained turbidity model was significant (p < 0.0001), with R2adj of 0.95 and RMSE of 25.0 FNU. The main effect and secondary effect of relative centrifugal force, the main effect of flow rate and the interaction of relative centrifugal force and flow rate were identified as significant factors. Flow rate had the greatest impact on centrate turbidity. In general, when RCF was less than 8000g, centrate turbidity only changed with flow rate.

[0396] Such centrifuges can then be collected for further processing, or filtered online via one or more depth filters to further clarify the sample. The most commonly used depth filters for bioprocessing are composed of diatomaceous earth, cellulose fibers, and positively charged resin binders. Unlike membrane filters, depth filters retain particles throughout the porous filter medium, allowing particles greater than and less than the pore size to be retained. It is believed that particle retention involves both size exclusion and adsorption via ions, hydrophobicity, and other mechanisms. Filter fouling mechanisms may include pore blockage, filter cake formation, and pore contraction. Non-limiting examples of depth filters that can be used in the context of the present disclosure include Millistak+X0HC, FOHC, DOHC, COHC, A1HC, B1HC depth filters (EMD Millipore), 3M TM Model 30 / 60ZA, 60 / 90ZA, VR05, VR07, Degrease Depth Filter (3M Corp.). 0.2μm filter, such as Sartorius 0.45 / 0.2μm Sartopore TM Dual-layer or Millipore SHR or SHC filter cassettes are usually placed after the depth filter. Emphaze AEX Hybrid Purifier Multi-Mechanism Filters can also be used to filter the depth filtered material. Other filters known to those skilled in the art can also be used.

[0397] After depth filtration, fine filtration is used to ensure final solids are removed prior to column chromatography. Fine filtration may include specially designed filters that increase impurity absorption via an ionic mechanism and thus reduce the overall impurity content applied to the primary column chromatography step. Guard filtration after fine filtration is only used for bioburden control.

[0398] To accommodate high titers and increased protein production, the filter pore size for these fine and guard filters can range from 0.15 μm to 0.25 μm, and in one aspect can be 0.2 μm. For an average 10,000 L bioreactor culture, the fine filter loading can be 255 L / m 2 Up to 270L / m 2 range, and in one aspect, about 265 L / m 2 Up to 270L / m 2 (For example, 33.6m 2 / 10,000L batch). Protection filter loading can be 700L / m 2 Up to 720L / m 2 within the range.

[0399] Protein A chromatography

[0400] In certain exemplary embodiments, it may be advantageous to subject a biological sample to affinity chromatography to produce a protein of interest. The chromatographic material is capable of selectively or specifically binding to or interacting with a protein of interest. Non-limiting examples of such chromatographic materials include: Protein A and Protein G. Also included are chromatographic materials comprising, for example, a protein or portion thereof that is capable of binding to or interacting with a protein of interest.

[0401] Affinity chromatography can involve subjecting the biological sample to a column containing a suitable Protein A resin. As used herein, the term "Protein A" encompasses Protein A recovered from its natural source, Protein A produced synthetically (e.g., by peptide synthesis or by recombinant techniques), and Protein A that retains and binds to proteins with C H 2 / C H Protein A can additionally bind to human IgG molecules containing IgG F(ab')2 fragments from the human VH3 gene family (Roben et al., Journal of Immunology 1995 154(12):6437-45). In certain aspects, Protein A resins are suitable for generating and separating a variety of antibody isotypes based on affinity by specifically interacting with the Fc portion of the molecule (if it has the region).

[0402] There are several commercial sources of protein A resins. Suitable resins include, but are not limited to, MabSelect PrismA, MabSelect SuRe, MabSelect SuRe LX, MabSelect, MabSelect SuRepcc, MabSelect Xtra, rProtein A Sepharose from Cytiva; ProSep HC, ProSepUltra, ProSep Ultra Plus from EMD Millipore; MabCapture from ThermoFisher; and Amsphere from JSR Life Sciences. TM A3.

[0403] Before the sample is loaded, the affinity column can be balanced with a suitable buffer. The pH of the protein A loading can be, for example, between about 6 and about 8, between about 6 and about 7, between about 7 and about 8, or about 6. After the column is loaded, the column can be washed once or multiple times using a suitable washing buffer. The column can then be eluted using an appropriate elution buffer (e.g., glycine-HCl, acetic acid, or citric acid). The eluent can be monitored using techniques well known to those skilled in the art, such as a UV detector. The eluted portion of interest can be collected and then prepared for further processing.

[0404] Protein A washing buffer can be selected based on its ability to destroy protein-protein interactions (e.g., interactions between proteins of interest and impurities such as HCPs) without destroying interactions between proteins of interest and chromatographic materials. Washing buffers suitable for removing HCPs may include, for example, salts (e.g., sodium salts such as sodium phosphate; potassium salts such as potassium sorbate; magnesium salts; hydrochloride-containing salts such as guanidine hydrochloride, salts containing Tris), surfactants (e.g., polysorbate 20, polysorbate 80), polar materials (e.g., isopropanol, ethanol) or amino acids (e.g., arginine). The pH of washing buffer suitable for removing HCPs may be between about 5 and about 9, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9.

[0405] Can be for manufacturing process based on removing relevant specific HCP, for example, by selecting the pH of the washing buffer corresponding to the pI of the relevant HCP to select suitable washing buffer.Can be based on, for example, destroying the electrostatic interaction of the relevant HCP with the protein of interest, the hydrophobic interaction of the relevant HCP with the protein of interest or the two to select washing buffer.The specific properties of the relevant HCP can be used to inform the selection of the buffer components as shown in (for example) Table 8.HCP analysis can be used to identify the relevant HCP, for example, by using mass spectrometry to identify and quantify the HCP associated with the protein of interest.Can be based on ensuring the acceptable yield of the protein of interest by destroying the combination of the protein of interest with the chromatographic resin as little as possible to select suitable washing buffer.

[0406] Table 8. Exemplary buffer compositions and pH for disrupting protein-protein interactions during affinity capture

[0407]

[0408] Affinity wash buffers can be further selected based on their ability to remove HMW species of the protein of interest. A suitable wash buffer can be selected that causes a substantial reduction in HMW species while maintaining the yield of the protein of interest at an acceptable level.

[0409] The use of affinity wash buffer to reduce HCP content and / or HMW species can enable the use of fewer downstream chromatography steps to achieve the corresponding API quality, such as using a protein A column and an AEX column without a CEX column or a HIC column; or using a protein A column, followed by a mixed mode column and an AEX column, without a CEX column or a HIC column. If the impurity content that can be achieved using another column is not matched by optimization of a separate affinity capture wash buffer, additional specific gravity can be obtained by using a charged filter medium during the filtration step. It is usually performed after the viral inactivation step, during which several pH adjustments have been made and filtering for specific conditions is easily incorporated into the downstream process. Filter media with multiple removal mechanisms are commercially available, including cationic, anionic, hydrophobic, and mixed mode mechanisms. Because the charge density on the filter is generally low when compared to packed bed chromatography, operating the filter under conditions where the product of interest is not bound increases the possibility of impurity removal and high recovery.

[0410] Low pH maintenance

[0411] In some exemplary embodiments, a recovery process can also be the main point of reducing or deactivating the virus that may be present in the biological sample. During the recovery phase of production, any one or more of a variety of virus reduction / deactivation methods can be used, including heat inactivation (pasteurization), pH inactivation, buffer / cleaner treatment, UV and gamma ray irradiation and adding some chemical inactivators, such as β-propiolactone or for example as described in U.S. Patent No. 4,534,972, the entire teaching content of the patent is incorporated herein by reference. In another aspect, the virus interception filtration as a dedicated virus removal step can also be used and discussed below.

[0412] In those exemplary embodiments employing viral reduction / inactivation, in one aspect, the biological sample can be conditioned as needed for other production steps. For example, after low pH viral inactivation, the pH of the sample can be adjusted to about neutral pH, e.g., about 4.5 to about 8.5, before continuing the production process. In another aspect, the mixture can also be diluted with water for injection (WFI) to obtain the desired conductivity.

[0413] In another aspect, the eluate can be subjected to viral inactivation, for example, caused by detergents or low pH. An appropriate detergent concentration or pH (and time) can be selected to obtain the desired viral inactivation results.

[0414] In another aspect, after viral inactivation, the pH and / or conductivity of the eluate can be adjusted for subsequent production steps, including, inter alia, anion exchange chromatography, cation exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., Protein A), and size exclusion chromatography.

[0415] Anion exchange chromatography

[0416] In certain exemplary embodiments, the protein of interest is produced by subjecting a biological sample to at least one anion exchange (AEX) separation step. In one case, the anion exchange step may be performed after an affinity chromatography procedure (e.g., protein A affinity). In other cases, the anion exchange step may be performed before the affinity chromatography step. In some embodiments, the AEX separation is the second of four chromatography unit operations and is performed downstream of affinity capture and viral inactivation performed by a low pH hold operation. In other embodiments, the AEX separation is preceded by an ion exchange step. Alternatively, the AEX separation may be followed by another ion exchange procedure.

[0417] Anion exchange packed bed chromatography is based on ionic interactions between the binding entity (target protein or impurity) and the functional groups immobilized on the chromatographic medium. Performance can vary with the mobile phase, functional groups, and resin backbone. In some embodiments, the specific goal of the AEX step is to reduce the content of DNA, HCCP, HMW species, and viruses (if present).

[0418] The use of anion exchange materials versus cation exchange materials is based in part on the local charge of the protein of interest.Anion exchange chromatography can be used in combination with other chromatography procedures such as affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and other chromatography modes known to those skilled in the art.

[0419] In carrying out the separation, the initial protein composition (biological sample) can be contacted with an anion exchange material using any of a variety of techniques, for example, using batch techniques or chromatographic techniques.

[0420] In the case of batch production, the anion exchange material is prepared in or equilibrated with the desired starting buffer. After preparation, a slurry of the anion exchange material can be obtained. The biological sample can be contacted with the slurry to allow the protein to adsorb to the anion exchange material. A solution containing acidic substances that do not bind to the AEX material can be separated from the slurry by allowing the slurry to settle and removing the supernatant. The slurry can be subjected to one or more washing steps and / or elution steps.

[0421] In the case of chromatographic separation, a packed bed chromatographic column is used to hold the chromatographic support material (resin or solid phase). The sample containing the protein of interest is loaded onto a specific chromatographic column. The column can then be subjected to one or more washing steps using a suitable washing buffer. Components of the sample that have not yet been adsorbed on the resin will likely flow through the column. Components that have been adsorbed on the resin can be eluted using a suitable elution buffer.

[0422] In some embodiments, the amount of protein loaded onto the AEX resin (e.g., grams of protein per liter of resin) is between about 50 g / L and about 200 g / L, between about 100 g / L and about 150 g / L, less than about 120 g / L, about 50 g / L, about 55 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, about 80 g / L, about 85 g / L, about 90 g / L, about 95 g / L, about 100 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, about 200 g / L, about 210 g / L, about 220 g / L, about 230 g / L, about 240 g / L, about 250 g / L, about 260 g / L, about 270 g / L, about 280 g / L, about 290 g / L, about 300 g / L, about 310 g / L, about 320 g / L g / L, about 170 g / L, about 175 g / L, about 180 g / L, about 185 g / L, about 190 g / L, about 195 g / L, or about 200 g / L.

[0423] In some embodiments, the sample can be neutralized prior to contacting with the AEX material. The sample, e.g., a virus inactivated pool, can be neutralized to a pH between about 7.40 and about 8.30, between about 7.50 and about 7.70, between about 7.55 and about 7.65, about 7.40, about 7.45, about 7.50, about 7.51, about 7.52, about 7.53, about 7.54, about 7.55, about 7.56, about 7.57, about 7.58, about 7.59 , about 7.60, about 7.61, about 7.62, about 7.63, about 7.64, about 7.65, about 7.66, about 7.67, about 7.68, about 7.69, about 7.70, about 7.75, about 7.80, about 7.85, about 7.90, about 7.95, about 8.00, about 8.05, about 8.10, about 8.15, about 8.20, about 8.25 or about 8.30.

[0424] In some embodiments, the sample can be further conditioned after neutralization and prior to contacting the AEX material. The sample, e.g., a virus inactivated pool, can be conditioned to between about 3.00 mS / cm and about 6.00 mS / cm, between about 3.00 mS / cm and about 4.00 mS / cm, between about 3.40 mS / cm and about 3.60 mS / cm, about 3.00 mS / cm, about 3.10 mS / cm, about 3.20 mS / cm, about 3.30 mS / cm, about 3.40 mS / cm, about 3.50 mS / cm, about 3.60 mS / cm, about 3.70 mS / cm, about 3.80 mS / cm, about 3.90 mS / cm, about 4.00 mS / cm, about 4.10 mS / cm, about 4.20 mS / cm, about 4.30 mS / cm, about 4.40 mS / cm, about 4.50 mS / cm, about 4.60 mS / cm, about 4.70 mS / cm, about 4.80 mS / cm, about 4.90 mS / cm, about 5.00 mS / cm, about 5.10 mS / cm, about 5.20 mS / cm, about 5.30 mS / cm, about 5.40 mS / cm The sample may be conditioned using, for example, about 2 M sodium acetate, about 2 M Tris base, or a combination thereof.

[0425] In some embodiments, the AEX column is subjected to a pre-equilibration step prior to contact with the sample. The pre-equilibration step displaces strongly bound hydroxide ions on the AEX column and promotes faster equilibrium. In some exemplary embodiments, the pre-equilibration buffer (or "mobile phase") comprises about 2M sodium chloride, WFI, or a combination thereof. In some exemplary embodiments, the linear velocity of the pre-equilibration buffer is between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, about 100 cm / hr, about 110 cm / hr, about 120 cm / hr, about 130 cm / hr, about 140 cm / hr, about 150 cm / hr, about 160 cm / hr, about 170 cm / hr, about 180 cm / hr, about 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, or about 300 cm / hr. In some exemplary embodiments, about two column volumes of pre-equilibration buffer are used.

[0426] In some embodiments, the AEX column is subjected to an equilibration step prior to contact with the sample and optionally after the pre-equilibration step. The equilibration step changes the pH and conductivity of the mobile phase to match the buffer excipients present in the load material (sample). In some embodiments, the equilibration buffer may comprise about 50 mM Tris and about 60 mM acetate, having a pH between about 7.50 and about 7.70 and a conductivity between about 3.00 mS / cm and about 4.00 mS / cm. In some embodiments, the pH of the equilibration buffer is between about 7.40 and about 8.30, between about 7.50 and about 7.70, between about 7.55 and about 7.65, about 7.40, about 7.45, about 7.50, about 7.51, about 7.52, about 7.53, about 7.54, about 7.55, about 7.56, about 7.57, about 7.58, about 7.59, about about 7.60, about 7.61, about 7.62, about 7.63, about 7.64, about 7.65, about 7.66, about 7.67, about 7.68, about 7.69, about 7.70, about 7.75, about 7.80, about 7.85, about 7.90, about 7.95, about 8.00, about 8.05, about 8.10, about 8.15, about 8.20, about 8.25 or about 8.30. In some embodiments, the conductivity of the equilibration buffer is between about 3.00 mS / cm and about 6.00 mS / cm, between about 3.00 mS / cm and about 4.00 mS / cm, between about 3.40 mS / cm and about 3.60 mS / cm, about 3.00 mS / cm, about 3.10 mS / cm, about 3.20 mS / cm, about 3.30 mS / cm, about 3.40 mS / cm, about 3.50 mS / cm, about 3.60 mS / cm, about 3.70 mS / cm, about 3.80 mS / cm, about 3.90 mS / cm, about 4.00 mS / cm, about 4.10 mS / cm, about 4.20 mS / cm, about 4.30 mS / cm, about 4.40 mS / cm, about 4.50 mS / cm, about 4.60 mS / cm, about 4.70 mS / cm, about 4.80 mS / cm, about 4.90 mS / cm, about 5.10 mS / cm, about 5.15 mS / cm, about 5.20 mS / cm, about 5.30 mS / cm, about 5.40 mS / cm .10mS / cm, about 4.20mS / cm, about 4.30mS / cm, about 4.40mS / cm, about 4.50mS / cm, about 4.60mS / cm, about 4.70mS / cm, about 4.80mS / cm, about 4.90mS / cm, about 5.00mS / cm, about 5.10mS / cm, about 5.20mS / cm, about 5.30mS / cm, about 5.40mS / cm, about 5.50mS / cm, about 5.60mS / cm, about 5.70mS / cm, about 5.80mS / cm, about 5.90mS / cm or about 6.00mS / cm.In some exemplary embodiments, the linear velocity of the equilibration buffer is between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, about 100 cm / hr, about 110 cm / hr, about 120 cm / hr, about 130 cm / hr, about 140 cm / hr, about 150 cm / hr, about 160 cm / hr, about 170 cm / hr, about 180 cm / hr, about 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, or about 300 cm / hr. {In some exemplary embodiments, about three column volumes of equilibration buffer are used.

[0427] In some embodiments, the concentration of protein loaded onto the AEX column ("AEX load," or grams of protein per liter of solution) is between about 10.0 g / L and about 30.0 g / L, between about 12 g / L and about 25 g / L, about 10.0 g / L, about 11.0 g / L, about 12.0 g / L, about 13.0 g / L, about 14.0 g / L, about 15.0 g / L , about 16.0 g / L, about 17.0 g / L, about 18.0 g / L, about 19.0 g / L, about 20.0 g / L, about 21.0 g / L, about 22.0 g / L, about 23.0 g / L, about 24.0 g / L, about 25.0 g / L, about 26.0 g / L, about 27.0 g / L, about 28.0 g / L, about 29.0 g / L or about 30.0 g / L. In some exemplary embodiments, the linear velocity of the AEX loading is between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, about 100 cm / hr, about 110 cm / hr, about 120 cm / hr, about 130 cm / hr, about 140 cm / hr, about 150 cm / hr, about 160 cm / hr, about 170 cm / hr, about 180 cm / hr, about 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, or about 300 cm / hr.

[0428] In some embodiments, when the UV absorbance at 280 nm reaches 0.2 AU on a 5 mm flow path, approximately 1.2 column volumes of flow-through are collected in the loading step. This is followed by a wash step to increase yield.

[0429] Typically in AEX chromatography, the washing step is performed using conditions similar to the loading conditions or alternatively by reducing the pH and / or increasing the ionic strength / conductivity of the washing solution in a stepwise or linear gradient manner. In one aspect, the saline solution used for the loading and washing buffers has a pH equal to or close to the isoelectric point (pi) of the protein of interest. Typically, the pH is about 0 to 2 units above or below the pI of the protein of interest, but it can be in the range of 0 to 0.5 units above or below. It can also be equal to the pI of the protein of interest.

[0430] In some embodiments, the wash buffer comprises about 50 mM Tris and about 60 mM acetate, has a pH between about 7.50 and about 7.70, and has a conductivity between about 3.00 mS / cm and about 4.00 mS / cm. In some embodiments, the wash buffer has a pH between about 7.40 and about 8.30, between about 7.50 and about 7.70, between about 7.55 and about 7.65, about 7.40, about 7.45, about 7.50, about 7.51, about 7.52, about 7.53, about 7.54, about 7.55, about 7.56, about 7.57, about 7.58, about 7.59, about about 7.60, about 7.61, about 7.62, about 7.63, about 7.64, about 7.65, about 7.66, about 7.67, about 7.68, about 7.69, about 7.70, about 7.75, about 7.80, about 7.85, about 7.90, about 7.95, about 8.00, about 8.05, about 8.10, about 8.15, about 8.20, about 8.25 or about 8.30. In some embodiments, the conductivity of the wash buffer is between about 3.00 mS / cm and about 6.00 mS / cm, between about 3.00 mS / cm and about 4.00 mS / cm, between about 3.40 mS / cm and about 3.60 mS / cm, about 3.00 mS / cm, about 3.10 mS / cm, about 3.20 mS / cm, about 3.30 mS / cm, about 3.40 mS / cm, about 3.50 mS / cm, about 3.60 mS / cm, about 3.70 mS / cm, about 3.80 mS / cm, about 3.90 mS / cm, about 4.00 mS / cm, about 4.10 mS / cm, about 4.20 mS / cm, about 4.30 mS / cm, about 4.40 mS / cm, about 4.50 mS / cm, about 4.60 mS / cm, about 4.70 mS / cm, about 4.80 mS / cm, about 4.90 mS / cm, about 5.00 mS / cm, about 5.10 mS / cm, about 5.20 mS / cm, about 5.30 mS / cm, about 5.40 mS / cm .10mS / cm, about 4.20mS / cm, about 4.30mS / cm, about 4.40mS / cm, about 4.50mS / cm, about 4.60mS / cm, about 4.70mS / cm, about 4.80mS / cm, about 4.90mS / cm, about 5.00mS / cm, about 5.10mS / cm, about 5.20mS / cm, about 5.30mS / cm, about 5.40mS / cm, about 5.50mS / cm, about 5.60mS / cm, about 5.70mS / cm, about 5.80mS / cm, about 5.90mS / cm or about 6.00mS / cm.In some embodiments, the linear velocity of the wash buffer is between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, about 100 cm / hr, about 110 cm / hr, about 120 cm / hr, about 130 cm / hr, about 140 cm / hr, about 150 cm / hr, about 160 cm / hr, about 170 cm / hr, about 180 cm / hr, about 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, or about 300 cm / hr. In some embodiments, the wash length is between about 3 column volumes (CV) and about 5 column volumes (CV), about 3 CV, about 3.5 CV, about 4 CV, about 4.5 CV, or about 5 CV.

[0431] In some embodiments, the AEX column can be regenerated using one or more stripping buffers. In some embodiments, the first stripping buffer comprises 2M sodium chloride (NaCl) and the second stripping buffer comprises 1N sodium hydroxide (NaOH). In some embodiments, the AEX column can be further soaked in 1N NaOH for at least about 30 minutes.

[0432] In some embodiments, the AEX resin life is between about 1 and about 100 cycles, between about 50 and about 90 cycles, less than about 100 cycles, about 10 cycles, about 20 cycles, about 40 cycles, about 50 cycles, about 60 cycles, about 70 cycles, about 80 cycles, about 90 cycles, or about 100 cycles. In some embodiments, the AEX resin life can reach 200 cycles.

[0433] The anionic reagent may be selected from the group consisting of acetate, chloride, formate and combinations thereof. The cationic reagent may be selected from the group consisting of Tris, arginine, sodium and combinations thereof. The buffer may be selected from the group consisting of pyridine, piperazine, L-histidine, Bis-Tris, Bis-Tris propane, imidazole, N-ethylmorpholine, TEA (triethanolamine), Tris, morpholine, N-methyldiethanolamine, AMPD (2-amino-2-methyl-1,3-propanediol), diethanolamine, ethanolamine, AMP (2-amino-2-methyl-1-propanol), 1,3-diaminopropane and piperidine.

[0434] The packed anion exchange chromatography column, anion exchange membrane device, anion exchange monomer device, or depth filtration medium can be operated in a bind-elute mode, a flow-through mode, or a mixed mode, wherein proteins exhibit binding to the chromatography material and can still be eluted from such material using a buffer that is the same as or substantially similar to the loading buffer.

[0435] In the binding-elution mode, the column or membrane device is first adjusted with a buffer having an appropriate ionic strength and pH under conditions where certain proteins will be adsorbed to a resin-based matrix. For example, during feed loading, the protein of interest may be adsorbed to the resin due to electrostatic attraction. After washing the column or membrane device with an equilibrium buffer or another buffer having a different pH and / or conductivity, product recovery is achieved by increasing the ionic strength (i.e., conductivity) of the elution buffer to compete with the solute for the charged sites of the anion exchange matrix. Changing the pH, thereby changing the charge of the solute, is another way to achieve solute elution. The change in conductivity or pH can be gradual (gradient elution) or stepwise (stepwise elution).

[0436] In flow-through mode, the column or membrane device is operated at a selected pH and conductivity so that the protein of interest does not bind to the resin or membrane, while the acidic substance will be retained on the column or will have an elution profile different from that of the protein of interest. In the case of this strategy, the acidic substance will interact with or bind to the chromatography material under suitable conditions, while the protein of interest and certain aggregates and / or fragments of the protein of interest will flow through the column.

[0437] In some embodiments, the AEX step is performed in negative mode (flow-through mode), where negatively charged process-related impurities are adsorbed to an immobilized positively charged ligand, and the protein of interest flows through.

[0438] Non-limiting examples of anion exchange resins include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amine (Q) groups. Other non-limiting examples include: Poros 50PI and Poros 50HQ, which are rigid polymeric beads with a backbone composed of cross-linked poly[styrene-divinylbenzene]; Poros 50XQ; Capto Q Impres and Capto DEAE, which are high flow agarose beads; Capto Adhere; Q Sepharose Fast Flow; Toyopearl QAE-550, Toyopearl DEAE-650, and Toyopearl GigaCap Q-650, which are polymeric basic beads; EMDTMAE Hicap, a synthetic polymeric resin with tentacle ion exchangers; Sartobind PA nano, which is a salt-tolerant chromatography membrane with primary amine ligands; Sartobind Q nano, which is a strong anion exchange chromatography membrane; CUNO BioCap, which is a ζ+ depth filtration medium composed of inorganic filter aids, refined cellulose, and ion exchange resins; XOHC, which is a depth filtration medium composed of inorganic filter aids, cellulose, and mixed cellulose esters; and Unosphere Q. In some embodiments, a resin with a relatively large pore size is selected for increased surface area exposed to negatively charged species.

[0439] In some embodiments, the sample (batch) may be split after the AEX step, wherein the split batches are processed simultaneously or sequentially. In some embodiments, the batch may not be lysed after the AEX step.

[0440] Additives such as polyethylene glycol (PEG), detergents, amino acids, sugars, chaotropes, etc. can be added to enhance separation performance, thereby achieving better separation, recovery and / or product quality.

[0441] Cation exchange chromatography

[0442] The method may include performing at least one cation exchange (CEX) step on a biological sample containing a protein of interest. In certain exemplary embodiments, the CEX step is performed in addition to the AEX step and before or after the AEX step. In some embodiments, CEX is the third chromatography unit operation in the purification process.

[0443] When performing cation exchange, a sample containing a protein of interest can be contacted with a cation exchange material using any of a variety of techniques, such as batch production techniques or chromatographic techniques, as described above for AEX. Cation exchange packed bed chromatography is based on ionic interactions between binding entities (target protein or impurities) and functional groups immobilized on a chromatographic medium. Performance can vary with, for example, mobile phase, elution conditions, functional groups, and resin backbone. In some embodiments, a specific goal of the CEX step is to reduce the content of HCCP and HMW product-related impurities, such as protein aggregates.

[0444] In some embodiments, the concentration of protein loaded onto the CEX resin (e.g., grams of protein per liter of resin) is between about 40 g / L and about 110 g / L, less than about 100 g / L, about 40 g / L, about 45 g / L, about 50 g / L, about 55 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, about 80 g / L, about 85 g / L, about 90 g / L, about 95 g / L, about 100 g / L, about 105 g / L, or about 110 g / L.

[0445] In some embodiments, a pre-stripping step is performed on the CEX column to remove any bound impurities before starting a new separation. In some embodiments, the pre-stripping buffer comprises about 2M sodium chloride (NaCl). In some embodiments, about two column volumes of pre-stripping buffer are used. In some embodiments, the pre-stripping step is only used for the first cycle of the batch.

[0446] In some embodiments, the CEX column is subjected to an equilibration step to change the mobile phase pH and conductivity to facilitate adsorption. In some embodiments, the equilibration buffer comprises about 40 mM sodium acetate, has a pH between about 5.90 and about 6.10, and a conductivity between about 2.00 mS / cm and about 4.00 mS / cm. In some embodiments, the equilibration buffer comprises a pH of between about 4.00 and about 6.50, between about 5.00 and about 6.00, between about 5.90 and about 6.10, about 4.00, about 4.10, about 4.20, about 4.30, about 4.40, about 4.50, about 4.60, about 4.70, about 4.80, about 4.90, about 5.00, about 5.10, about 5.20, about 5.30, about 5.40, about 5.50, about 5.60, about 5.70, about 5.80, about 5.90, about 6.00, about 6.10, about 6.20, about 6.30, about 6.40, or about 6.50. In some embodiments, the conductivity of the equilibration buffer is between about 2.00 mS / cm and about 6.00 mS / cm, between about 2.00 mS / cm and about 4.00 mS / cm, between about 4.00 mS / cm and about 6.00 mS / cm, between about 3.00 mS / cm and about 4.00 mS / cm, about 2.00 mS / cm, about 2.10 mS / cm, about 2. 20mS / cm, about 2.30mS / cm, about 2.40mS / cm, about 2.50mS / cm, about 2.60mS / cm, about 2.70mS / cm, about 2.80mS / cm, about 2.90mS / cm, about 3.00mS / cm, about 3.10mS / cm, about 3.20mS / cm, about 3.30mS / cm, about 3.40mS / cm, about 3 .50mS / cm, about 3.60mS / cm, about 3.70mS / cm, about 3.80mS / cm, about 3.90mS / cm, about 4.00mS / cm, about 4.10mS / cm, about 4.20mS / cm, about 4.30mS / cm, about 4.40mS / cm, about 4.50mS / cm, about 4.60mS / cm, about 4.70mS / cm, about In some embodiments, about three column volumes of equilibration buffer are used.

[0447] The sample can be conditioned prior to contact with the CEX material (CEX loading). In some embodiments, the sample (e.g., AEX pool) is adjusted to a pH between about 4.00 and about 6.50, between about 5.00 and about 6.00, between about 5.90 and about 6.10, about 4.00, about 4.10, about 4.20, about 4.30, about 4.40, about 4.50, about 4.60, about 4.70, about 4.80, about 4.90, about 5.00, about 5.10, about 5.20, about 5.30, about 5.40, about 5.50, about 5.60, about 5.70, about 5.80, about 5.90, about 6.00, about 6.10, about 6.20, about 6.30, about 6.40, or about 6.50. In some embodiments, the sample is conditioned using about 2 M acetic acid.

[0448] In some embodiments, a wash step is performed on the CEX column to remove weakly bound impurities. In some embodiments, the equilibration buffer also serves as a wash buffer. In some embodiments, the wash buffer comprises about 40 mM sodium acetate, has a pH between about 5.90 and about 6.10, and has a conductivity between about 2.00 mS / cm and about 4.00 mS / cm. In some embodiments, the wash buffer comprises a pH of between about 4.00 and about 6.50, between about 5.00 and about 6.00, between about 5.90 and about 6.10, about 4.00, about 4.10, about 4.20, about 4.30, about 4.40, about 4.50, about 4.60, about 4.70, about 4.80, about 4.90, about 5.00, about 5.10, about 5.20, about 5.30, about 5.40, about 5.50, about 5.60, about 5.70, about 5.80, about 5.90, about 6.00, about 6.10, about 6.20, about 6.30, about 6.40, or about 6.50. In some embodiments, the conductivity of the wash buffer is between about 2.00 mS / cm and about 6.00 mS / cm, between about 2.00 mS / cm and about 4.00 mS / cm, between about 4.00 mS / cm and about 6.00 mS / cm, between about 3.00 mS / cm and about 4.00 mS / cm, about 2.00 mS / cm, about 2.10 mS / cm, about 2. 20mS / cm, about 2.30mS / cm, about 2.40mS / cm, about 2.50mS / cm, about 2.60mS / cm, about 2.70mS / cm, about 2.80mS / cm, about 2.90mS / cm, about 3.00mS / cm, about 3.10mS / cm, about 3.20mS / cm, about 3.30mS / cm, about 3.40mS / cm, about 3 .50mS / cm, about 3.60mS / cm, about 3.70mS / cm, about 3.80mS / cm, about 3.90mS / cm, about 4.00mS / cm, about 4.10mS / cm, about 4.20mS / cm, about 4.30mS / cm, about 4.40mS / cm, about 4.50mS / cm, about 4.60mS / cm, about 4.70mS / cm, about In some embodiments, the wash buffer comprises potassium sorbate.

[0449] After the washing step, the protein of interest can be eluted via increased conductivity. In some embodiments, CEX pool collection starts at 0.2AU using a 2mm UV path and ends after about 5 column volumes (CV) of pool collection. In some embodiments, the elution buffer comprises about 20mM Tris and about 120mM sodium acetate, with a pH between about 5.90 and about 6.20, and a conductivity between about 9.00mS / cm and about 11.00mS / cm. In some embodiments, the elution buffer comprises a pH between about 5.70 and about 7.00, between about 5.90 and about 6.20, about 5.70, about 5.80, about 5.90, about 6.00, about 6.10, about 6.20, about 6.30, about 6.40, about 6.50, about 6.60, about 6.70, about 6.80, about 6.90, or about 7.00. In some embodiments, the conductivity of the equilibration buffer is between about 7.00 mS / cm and about 11.00 mS / cm, between about 9.00 mS / cm and about 11.00 mS / cm, between about 10.00 mS / cm and about 11.00 mS / cm, about 7.00 mS / cm, about 7.10 mS / cm, about 7.20 mS / cm, about 7.30 mS / cm, about 7.40 mS / cm, about 7.60 mS / cm, about 7.70 mS / cm, about 7.80 mS / cm, about 7.90 mS / cm, about 8.10 mS / cm, about 8.20 mS / cm, about 8.30 mS / cm, about 8.40 mS / cm, about 8.60 mS / cm, about 8.70 mS / cm, about 8.80 mS / cm, about 8.90 mS / cm, about 8.10 mS / cm, about 8.20 mS / cm, about 8.30 mS / cm, about 8.40 mS / cm, about 8.50 mS / cm, about 8.60 mS / cm, about 8.70 mS / cm, about 8.80 mS / cm, about 8.90 mS / cm, about 8. m, about 7.40mS / cm, about 7.50mS / cm, about 7.60mS / cm, about 7.70mS / cm, about 7.80mS / cm, about 7.90mS / cm, about 8.00mS / cm, about 8.10mS / cm, about 8.20mS / cm, about 8.30mS / cm, about 8.40mS / cm, about 8.50mS / cm, about 8.60m S / cm, about 8.70mS / cm, about 8.80mS / cm, about 8.90mS / cm, about 9.00mS / cm, about 9.10mS / cm, about 9.20mS / cm, about 9.30mS / cm, about 9.40mS / cm, about 9.50mS / cm, about 9.60mS / cm, about 9.70mS / cm, about 9.80mS / cm, about 9. In some embodiments, about five column volumes of elution buffer are used.

[0450] In some embodiments, after elution, the CEX column is subjected to high ionic strength stripping followed by caustic stripping to remove bound impurities and prepare the resin for additional cycles or storage. In some embodiments, the first stripping buffer comprises about 2M sodium chloride (NaCl) and the second stripping buffer comprises about 1N sodium hydroxide (NaOH).

[0451] In some embodiments, each stage of the separation except elution is performed at a linear velocity of between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, about 100 cm / hr, about 110 cm / hr, about 120 cm / hr, about 130 cm / hr, about 140 cm / hr, about 150 cm / hr, about 160 cm / hr, about 170 cm / hr, about 180 cm / hr, about 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, or about 300 cm / hr. In an exemplary embodiment, each stage of the separation except elution is performed at a linear velocity of 200 cm / hr. In an exemplary embodiment, the elution step is performed at a linear velocity of 100 cm / hr.

[0452] In some embodiments, the CEX resin life is between about 1 and about 100 cycles, between about 50 and about 90 cycles, less than about 100 cycles, about 10 cycles, about 20 cycles, about 30 cycles, about 40 cycles, about 50 cycles, about 60 cycles, about 70 cycles, about 80 cycles, about 90 cycles, or about 100 cycles. In some embodiments, the AEX resin life can reach 200 cycles.

[0453] Saline solutions can be used as both loading and washing buffers at a pH below the isoelectric point (pi) of the protein of interest. In one aspect, the pH is about 0 to 5 units lower than the pi of the protein. In another aspect, it is in the range of 1 to 2 units lower than the pi of the protein. In yet another aspect, it is in the range of 1 to 1.5 units lower than the pi of the protein.

[0454] In certain exemplary embodiments, the concentration of the anionic agent in the aqueous solution is increased or decreased to achieve a pH between about 3.5 and about 10.5, or between about 4 and about 10, or between about 4.5 and about 9.5, or between about 5 and about 9, or between about 5.5 and about 8.5, or between about 6 and about 8, or between about 6.5 and about 7.5. In one aspect, the concentration of the anionic agent in the saline solution is increased or decreased to achieve a pH of 5, or 5.5, or 6, or 6.5, or 6.8, or 7.5. Buffer systems suitable for CEX methods include, but are not limited to, Tris formate, Tris acetate, ammonium sulfate, sodium chloride, and sodium sulfate.

[0455] In certain exemplary embodiments, the conductivity and pH of the saline solution are adjusted by increasing or decreasing the concentration of a cationic agent. In one aspect, the concentration of the cationic agent is maintained in the range of about 20 mM to about 500 mM, about 50 mM to about 350 mM, about 100 mM to about 300 mM, or about 100 mM to about 200 mM. Non-limiting examples of cationic agents can be selected from the group consisting of sodium, Tris, triethylamine, ammonium, arginine, and combinations thereof.

[0456] The packed cation exchange chromatography column or cation exchange membrane device can be operated in bind-elute mode, flow-through mode, or mixed mode, in which the product exhibits binding or interaction with the chromatographic material, but can still be eluted from such material using a buffer that is the same as or substantially similar to the loading buffer (the details of these modes are outlined above). In some embodiments, the CEX step is performed in a positive (bind-elute) mode, in which the positively charged protein of interest and impurities are adsorbed to a fixed negatively charged stationary phase. The protein of interest is subsequently eluted, while many impurities remain bound to the stationary phase. A series of regeneration steps remove bound impurities and prepare the CEX column for subsequent cycles.

[0457] Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S). Additional cationic materials include, but are not limited to: Capto SPImpRes, which are high flow agarose beads; CaptoS ImpAct; F-grade CM Hyper D, which are coated ceramic beads and impregnated with functionalized hydrogel (250 to 400 ionic groups μeq / mL); Eshmuno S, which is a hydrophilic polyvinyl ether basic matrix with 50 to 100 μM / mL ionic capacity; Nuvia C Prime, which is a hydrophobic cation exchange medium composed of a 55 to 75 με / mL macroporous, highly cross-linked, hydrophilic polymer matrix; Nuvia S, which has a UNOspher basic matrix with 90 to 150 με / mL ionic groups; Poros HS, which are rigid polymeric beads with a backbone composed of cross-linked poly[styrene-divinylbenzene]; Poros XS, which are rigid polymeric beads with a backbone composed of cross-linked poly[styrene-divinylbenzene]; Toyo Pearl Giga Cap CM Toyo Pearl Giga Cap S650M, which are polymeric basic beads with an ionic capacity of 0.225 meq / mL; Toyo Pearl Giga Cap S650M, which are polymeric basic beads; Toyo Pearl MX TRP, which are polymeric basic beads; and EMD SE Hicap. It should be noted that CEX chromatography can be used with the MM resins described herein. In some embodiments, a CEX resin with a relatively high capacity is selected, for example, for loading up to about 100 g / L of resin. In some embodiments, a CEX resin with a relatively high caustic stability is selected, for example, to prevent hydrolysis of cationic substrates.

[0458] Additives such as polyethylene glycol, detergents, amino acids, sugars, and chaotropes can be added to enhance separation performance, resulting in better separation, recovery, and / or product quality.

[0459] Mixed Mode Chromatography

[0460] Mixed mode ("MM") chromatography can be used in the process of the present invention. MM chromatography (also referred to herein as "multimodal chromatography" or "MMC") is a chromatography strategy using a support comprising a ligand that is capable of providing at least two different interactions with an analyte or protein of interest from a sample. One of these sites provides an attractive type of charge interaction between the ligand and the protein of interest, and another site provides an electron acceptor-donor interaction and / or a hydrophobic and / or hydrophilic interaction. Electron donor-acceptor interactions include interactions such as hydrogen bonding, π-π, cation-π, charge transfer, dipole-dipole, induced dipole, and the like.

[0461] The column resin for mixed mode separation can be Capto Adhere. Capto Adhere is a strong anion exchanger with multi-mode functionality. Its alkaline matrix is ​​a highly cross-linked agarose with a ligand (N-benzyl-N-methylethanolamine), which exhibits different functionalities for interaction, such as ionic interaction, hydrogen bonding and hydrophobic interaction. In certain aspects, the resin for mixed mode separation is selected from PPA-HyperCel and HEA-HyperCel. The alkaline matrix of PPA-HyperCel and HEA-HyperCel is a high porosity cross-linked cellulose. Its ligands are amphetamine and hexylamine, respectively. Amphetamine and hexylamine provide different selectivities and hydrophobicity selections for protein separation. Other mixed mode chromatographic carriers include, but are not limited to, Capto MMC, MEP-HyperCel, MBI HyperCel, CMM HyperCel, Capto AdhereImpRes, Capto Core 700, Nuvia C Prime, Toyo Pearl MX Trp 650M and HCX. In some aspects, the mixed mode chromatography resin comprises a ligand coupled to an organic or inorganic carrier, sometimes indicated directly by a basic matrix or via a spacer. The carrier may be in the form of particles, such as substantially spherical particles, monomers, filters, films, surfaces, capillaries, etc. In some aspects, the carrier is prepared by a natural polymer, such as a cross-linked carbohydrate material, such as agarose, agar, cellulose, polydextrose, polyglucosamine, konjac, carrageenan, gellan gum, alginate, etc. In order to obtain a high adsorption capacity, the carrier may be porous, and the ligand is then coupled to the outer surface and to the pore surface. Such natural polymer carriers can be prepared according to standard methods, such as reverse suspension gelation (S Hjerten: Biochemistry and Biophysics Acta (BiochimBiophys Acta) 79 (2), 393-398 (1964), all of which are incorporated herein by reference). Alternatively, the support may be prepared from a synthetic polymer, such as a cross-linked synthetic polymer, for example styrene or styrene derivatives, divinylbenzene, acrylamide, acrylate, methacrylate, vinyl ester, vinyl amide, etc. Such synthetic polymers may be produced according to standard methods, see "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimicae L'Industria 70(9), 70-75 (1988), the entire teaching of which is incorporated herein by reference). Porous natural or synthetic polymer supports may also be purchased from commercial sources, such as Cytiva, Uppsala, Sweden. The MMC may be operated in flow-through mode or bind and elute mode depending on the resin used; for example, an AEX / HIC hybrid resin may preferably be operated in flow-through mode, while a CEX / HIC hybrid resin may preferably be operated in bind and elute mode.

[0462] Additives such as polyethylene glycol, detergents, amino acids, sugars, chaotropes, etc. can be added to enhance separation performance, thereby obtaining better separation, recovery and / or product quality.

[0463] The methods of the present disclosure can also be implemented in a continuous chromatography mode. In this mode, at least two columns (referred to as a "first" column and a "second" column) are used. In certain embodiments, this continuous chromatography mode can be performed so that the eluted fractions and / or the stripped fractions can be subsequently or simultaneously loaded onto the second column (with or without dilution).

[0464] In one embodiment, the medium of choice for the continuous mode may be one of various chromatography resins, monomeric media, membrane adsorbent media, or depth filtration media having pendant hydrophobic and anion exchange functional groups.

[0465] In some exemplary embodiments, MMC is the second chromatographic separation after affinity capture chromatography and optionally before the AEX step in the process. In other embodiments, MMC is the third chromatographic separation after affinity capture chromatography and AEX steps in the process. In some embodiments, the method of the present disclosure may include an MMC step instead of a CEX step or a HIC step. In some embodiments, the process for producing a recombinant protein (such as Dupilumab) includes two, three or four chromatographic modes, including affinity capture chromatography, MMC and optional AEX, CEX, HIC or another MMC step in any order before or after MMC. Regardless of the order of the chromatographic steps, MMC can be operated in a flow-through mode or in a binding and elution mode. MMC can be operated based on a culture plate format (e.g., a 96-well plate format or a robotic column chromatography format).

[0466] In some embodiments, the MMC column is subjected to an equilibration step to change the mobile phase pH and conductivity to facilitate adsorption. In some embodiments, the equilibration buffer comprises about 100 mM NaCl at a pH of about 5. In some embodiments, the equilibration buffer comprises between about 4.50 and about 9.00, between about 4.50 and about 8.00, between about 4.50 and about 5.50, between about 5.00 and about 6.00, about 4.50, about 4.60, about 4.70, about 4.80, about 4.90, about 5.00, about 5.10, about 5.20, about 5.30, about 5.40, about 5.50, about 5.60, about 5.70, about 5.80, about 5.90, about 6.00, about 6.10 , about 6.20, about 6.30, about 6.40, about 6.50, about 6.60, about 6.70, about 6.80, about 6.90, about 7.00, about 7.10, about 7.20, about 7.30, about 7.40, about 7.50, about 7.60, about 7.70, about 7.80, about 7.90, about 8.00, about 8.10, about 8.20, about 8.30, about 8.40, about 8.50, about 8.60, about 8.70, about 8.80, about 8.90 or about 9.00.

[0467] In some embodiments, the equilibration buffer comprises between about 100 mM and about 500 mM, between about 100 mM and about 250 mM, between about 100 mM and about 150 mM, between about 80 mM and about 120 mM, between about 95 mM and about 105 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM. In some embodiments, the equilibration buffer comprises about 0 mM to about 100 mM NaCl. In some embodiments, the equilibration buffer comprises citrate or arginine.

[0468] In some embodiments, the wash buffer may comprise any of the pH value, NaCl, concentration, or buffer salt of the equilibration buffer described above.

[0469] In some embodiments, the amount of protein loaded on the MMC resin (e.g., grams of protein per liter of resin) is about 100 g / L or about 110 g / L. In some embodiments, the amount of protein loaded on the MMC resin is between about 50 g / L and about 200 g / L, between about 100 g / L and about 150 g / L, between about 100 g / L and about 110 g / L, less than about 120 g / L, about 50 g / L, about 55 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, about 80 g / L, about 85 g / L, about 90 g / L, about 95 g / L, about 10 ... g / L, about 170 g / L, about 175 g / L, about 180 g / L, about 185 g / L, about 190 g / L, about 195 g / L, or about 200 g / L. In some embodiments, the amount of protein loaded on the MMC resin can be between about 10 g / L and about 80 g / L, about 10 g / L, about 15 g / L, about 20 g / L, about 25 g / L, about 30 g / L, about 35 g / L, about 40 g / L, about 45 g / L, about 50 g / L, about 55 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, or about 80 g / L.

[0470] In some embodiments, the MMC is operated in a bind and elute mode and further comprises using an elution buffer. In some embodiments, the elution buffer comprises between about 4.50 and about 9.00, between about 4.50 and about 8.00, between about 4.50 and about 5.50, between about 5.00 and about 6.00, about 4.50, about 4.60, about 4.70, about 4.80, about 4.90, about 5.00, about 5.10, about 5.20, about 5.30, about 5.40, about 5.50, about 5.60, about 5.70, about 5.80, about 5.90, about 6.00, about 6.10 , about 6.20, about 6.30, about 6.40, about 6.50, about 6.60, about 6.70, about 6.80, about 6.90, about 7.00, about 7.10, about 7.20, about 7.30, about 7.40, about 7.50, about 7.60, about 7.70, about 7.80, about 7.90, about 8.00, about 8.10, about 8.20, about 8.30, about 8.40, about 8.50, about 8.60, about 8.70, about 8.80, about 8.90 or about 9.00.

[0471] In some embodiments, the elution buffer comprises between about 0 mM and about 500 mM, between about 100 mM and about 250 mM, between about 100 mM and about 150 mM, about 0 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM. In some embodiments, the elution buffer comprises citrate or arginine.

[0472] Hydrophobic Interaction Chromatography

[0473] The methods of the present disclosure may include performing at least one hydrophobic interaction chromatography (HIC) step on a biological sample containing a protein of interest. When performing the separation, the biological sample is contacted with a HIC material, for example using batch production techniques or using column or membrane chromatography. Prior to the HIC treatment, it may be necessary to adjust the concentration of the salt buffer to achieve the desired protein binding / interaction with the resin or membrane. In some embodiments, the HIC separation is the fourth and final chromatographic separation in the process and is performed downstream of the CEX step.

[0474] In some embodiments, a specific goal of the HIC step can be, for example, to reduce the content of HCP or HMW product-related impurities, including PLBD2. The PLBD2 content in the HIC load can be present at 100-300 ppm, and can be reduced by about 40x, about 50x, about 60x, about 70x, about 80x, about 90x, about 100x, about 110x, about 120x, about 130x, about 140x, about 150x, about 160x, about 170x, about 180x, about 190x, about 200x, about 210x, about 220x, about 230x, about 240x, about 250x, about 260x, about 270x, about 280x, about 290x, about 300x, or about 310x, below 100 ppm, below 30 ppm, below 4 ppm, or below 1 ppm. The use of HIC to remove PLBD2 is also described in more detail in US Pat. No. 10,774,141, which is incorporated herein by reference in its entirety for all purposes.

[0475] In some embodiments, a batch of sample can be split into two sub-batches that are processed individually prior to reconstitution, such as prior to third stage concentration, via a HIC step and subsequent steps, such as virus retention filtration and UF / DF.

[0476] While ion exchange chromatography relies on the local charge of the protein of interest for selective separation, hydrophobic interaction chromatography exploits the hydrophobic properties of proteins to achieve selective separation. HIC resins are typically functionalized with aromatic or aliphatic hydrocarbon ligands. The hydrophobic groups on or within the protein interact with the hydrophobic groups of the chromatographic resin or membrane. Typically, under suitable conditions, the more hydrophobic a protein (protein moiety) is, the stronger its interaction with the column or membrane will be. Therefore, under suitable conditions, HIC can be used to facilitate the separation of process-related impurities (e.g., HCPs) and product-related substances (e.g., aggregates and fragments) from the protein of interest in a sample.

[0477] As with ion exchange chromatography, HIC columns or HIC membrane devices may also be operated in elution mode, flow-through, or mixed mode, in which the product exhibits binding or interaction with the chromatography material and may be washed from such material using a buffer that is the same as or substantially similar to the loading buffer. (Details of these modes are outlined above with respect to AEX processing.) In some embodiments, the HIC step is performed in negative mode, in which process-related impurities are bound to an immobilized ligand, and the protein of interest flows through.

[0478] Since hydrophobic interactions are strongest at high ionic strengths, this form of separation is preferably performed after a salt elution step such as those commonly used in conjunction with ion exchange chromatography. Alternatively, salt may be added to the sample prior to employing the HIC step. High salt concentrations favor adsorption of proteins to the HIC column, but actual concentrations may vary widely depending on the nature of the protein of interest, the type of salt, and the particular HIC ligand selected. The various ions may be arranged in a so-called solvophobic series depending on whether they promote hydrophobic interactions (salting out) or disrupt water structure (chaotropy) and result in a weakening of hydrophobic interactions. Cations are arranged according to increasing salting out as Ba, C, and D. 2+ , Ca 2+ Mg 2+ , Li + , Cs + 、Na + , K + , Rb + NH4 + , and the anions are expelled as PO according to the increasing chaotropic effect. 4 3- 、SO 4 2- , CH 3 CO 3 - , CI - Br - 、NO 3 - , ClO 4 - ,I - 、SCN - .

[0479] HIC media typically comprise a basic matrix (e.g., cross-linked agarose or a synthetic copolymer material) coupled to a hydrophobic ligand (e.g., an alkyl or aryl group). Suitable HIC media include agarose resins or membranes functionalized with phenyl groups (e.g., Phenyl Sepharose from Stovam). TMor Phenyl Membrane from Sartorius). Various HIC resins are commercially available. Examples include, but are not limited to, Capto Phenyl, Capto Butyl, low or high substituted fast flow phenyl Sepharose TM 6. High-efficiency phenyl Sepharose TM , Highly effective Octyl Sepharose TM (GE Healthcare); Fractogel TM EMD Propyl or Fractogel TM EMD Phenyl (E.Merck, Germany); Macro-Prep TM Methyl or Macro-Prep TM tert-Butyl column (Bio-Rad, California); WP HI-propyl (C3) TM (JT Baker, New Jersey); and Toyopearl TM ether, phenyl, or butyl (TosoHaas, PA); Toyo PPG; Toyo phenyl; Toyo butyl; and Toyo hexyl.

[0480] Since the pH selected for any particular production process must be compatible with protein stability and activity, specific pH conditions may be specific for each application. However, since at pH 5.0-8.5, the effect of specific pH values ​​on the final selectivity and resolution of HIC separation is minimal, such conditions may be advantageous. At pH values ​​above 8.5 or below 5.0, an increase in pH weakens hydrophobic interactions and changes protein retention to a greater extent. In addition, changes in ionic strength, the presence of organic solvents, temperature, and pH (especially at isoelectric pI, when there is no net surface charge) may affect protein structure and solubility, and therefore affect interactions with other hydrophobic surfaces (such as those in HIC culture medium), therefore, in certain embodiments, the present disclosure incorporates production strategies, wherein one or more of the aforementioned strategies are adjusted to achieve the desired reduction of program-related impurities and / or product-related substances.

[0481] In certain exemplary embodiments, spectroscopic methods such as UV, NIR, FTIR, fluorescence and Raman can be used to monitor the proteins and impurities of interest in an on-line, near-line or online mode, which can then be used to control the aggregate content in the pooled material collected from the HIC adsorbent effluent. In other exemplary embodiments, on-line, near-line or online monitoring methods can be used on the effluent pipeline of the chromatography step or in the collection container to enable the desired product quality / recovery to be achieved. In other exemplary embodiments, the UV signal can be used as a surrogate to achieve the appropriate product quality / recovery, wherein the UV signal can be appropriately processed, including but not limited to processing techniques such as integration, differentiation and moving average, so that common process variations can be resolved and the target product quality can be achieved. In certain exemplary embodiments, such measurements can be combined with an online dilution method so that the ion concentration / conductivity of the load / wash solution can be feedback controlled and thus promote product quality control.

[0482] In some embodiments, the concentration of protein loaded onto the HIC resin (e.g., grams of protein per liter of resin) is between about 60 g / L and about 180 g / L, between about 100 g / L and about 150 g / L, between about 180 g / L and about 200 g / L, less than about 180 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, about 80 g / L, about 85 g / L, about 90 g / L, about 95 g / L, about 100 g / L, or about 150 g / L. g / L, about 170 g / L, about 175 g / L, about 175 g / L, about 180 g / L, about 185 g / L, about 190 g / L, about 195 g / L, or about 200 g / L.

[0483] In some embodiments, the sample can be conditioned prior to contact with the HIC material, which can be referred to as loading conditioning. In some embodiments, a sample, such as a CEX pool, is conditioned to about 40 mM citrate using a single bolus injection addition of about 1.2 M sodium citrate. After conditioning, the pH of the sample (HIC loading) is between about 6.30 and about 6.70, and the conductivity of the sample is between about 14.00 and about 17.00 mS / cm. In some embodiments, the HIC loading comprises a pH between about 5.50 and about 7.00, between about 6.30 and about 6.70, about 5.50, about 5.60, about 5.70, about 5.80, about 5.90, about 6.00, about 6.10, about 6.20, about 6.30, about 6.40, about 6.50, about 6.60, about 6.70, about 6.80, about 6.90, or about 7.00. In some embodiments, the HIC loading comprises between about 14.00 mS / cm and about 17.00 mS / cm, about 14.00 mS / cm, about 14.10 mS / cm, about 14.20 mS / cm, about 14.30 mS / cm, about 14.40 mS / cm, about 14.50 mS / cm, about 14.60 mS / cm, about 14.70 mS / cm, about 14.80 mS / cm, about 14.90 mS / cm, about 15.00 mS / cm, about 15.10 mS / cm, about 15.20 mS / cm, about 15.30 mS / cm , about 15.40 mS / cm, about 15.50 mS / cm, about 15.60 mS / cm, about 15.70 mS / cm, about 15.80 mS / cm, about 15.90 mS / cm, about 16.00 mS / cm, about 16.10 mS / cm, about 16.20 mS / cm, about 16.30 mS / cm, about 16.40 mS / cm, about 16.50 mS / cm, about 16.60 mS / cm, about 16.70 mS / cm, about 16.80 mS / cm, about 16.90 mS / cm or about 17.00 mS / cm. In some embodiments, the concentration of citrate in the HIC load is between about 10 mM and about 50 mM, between about 30 mM and about 40 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 45 mM, or about 50 mM.

[0484] In some embodiments, the HIC column is subjected to a pre-equilibration step prior to contact with the sample. The pre-equilibration step removes all residual bound impurities from the column. In some embodiments, the pre-equilibration step is performed only for the first cycle of a batch. In some exemplary embodiments, the pre-equilibration buffer comprises water for injection (WFI). In some exemplary embodiments, the linear velocity of the pre-equilibration buffer is between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, about 100 cm / hr, about 110 cm / hr, about 120 cm / hr, about 130 cm / hr, about 140 cm / hr, about 150 cm / hr, about 160 cm / hr, about 170 cm / hr, about 180 cm / hr, about 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, or about 300 cm / hr. In an exemplary embodiment, the linear velocity is about 200 cm / hr. In some exemplary embodiments, about three column volumes of pre-equilibration buffer are used.

[0485] In some embodiments, the HIC column is subjected to an equilibration step prior to contact with the sample and optionally after the pre-equilibration step. The equilibration step changes the pH and conductivity of the mobile phase to match the buffer excipients present in the loading material (sample). In some embodiments, the equilibration buffer may comprise about 40 mM Tris and about 40 mM sodium citrate, with a pH between about 6.30 and about 6.70, and a conductivity between about 8.50 mS / cm and about 12.00 mS / cm. In some embodiments, the linear velocity of the equilibration buffer is between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, about 100 cm / hr, about 110 cm / hr, about 120 cm / hr, about 130 cm / hr, about 140 cm / hr, about 150 cm / hr, about 160 cm / hr, about 170 cm / hr, about 180 cm / hr, about 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, or about 300 cm / hr. In an exemplary embodiment, the linear velocity is about 200 cm / hr. In some exemplary embodiments, about two column volumes of equilibration buffer are used.

[0486] In some embodiments, the protein concentration loaded at the HIC is between about 10.0 g / L and about 20.0 g / L, between about 12.0 g / L and about 18.0 g / L, about 10.0 g / L, about 10.5 g / L, about 11.0 g / L, about 11.5 g / L, about 12.0 g / L, about 12.5 g / L, about 13.0 g / L, about 13.5 g / L, about 14.0 g / L, about 14.5 g / L, about 15.0 g / L, about 15.5 g / L, about 16.0 g / L, about 16.5 g / L, about 17.0 g / L, about 17.5 g / L, about 18.0 g / L, about 18.5 g / L, about 19.0 g / L, about 19.5 g / L, or about 20.0 g / L. In some embodiments, the linear speed of the HIC loading is between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, about 100 cm / hr, about 110 cm / hr, about 120 cm / hr, about 130 cm / hr, about 140 cm / hr, about 150 cm / hr, about 160 cm / hr, about 170 cm / hr, about 180 cm / hr, about 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, or about 300 cm / hr. In an exemplary embodiment, the linear speed is about 150 cm / hr.

[0487] In some embodiments, when the UV absorbance at 280 nm reaches 0.2 AU on a 5 mm flow path, approximately 3 to 4 column volumes of flow-through are collected in the loading step. This is followed by a wash step to increase yield.

[0488] In some embodiments, the equilibration buffer also serves as a wash buffer. In some embodiments, the wash buffer comprises about 40 mM Tris and about 40 mM sodium citrate, a pH between about 6.30 and about 6.70, and a conductivity between about 8.50 mS / cm and about 12.00 mS / cm. In some embodiments, the linear velocity of the wash buffer is between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, about 100 cm / hr, about 110 cm / hr, about 120 cm / hr, about 130 cm / hr, about 140 cm / hr, about 150 cm / hr, about 160 cm / hr, about 170 cm / hr, about 180 cm / hr, about 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, or about 300 cm / hr. In an exemplary embodiment, the linear velocity is about 150 cm / hr. In some embodiments, the wash length is between about 6 and about 8 column volumes (CV), about 6CV, about 6.5CV, about 7CV, about 7.5CV, or about 8CV. In an exemplary embodiment, the wash length is about 8CV.

[0489] In some embodiments, the HIC column can be regenerated using one or more stripping buffers. In some embodiments, the first stripping buffer comprises about two column volumes of WFI, the second stripping buffer comprises about two column volumes of 1N sodium hydroxide, and the third stripping buffer comprises about two column volumes of WFI. The stripping buffer may comprise 5mM sodium hydroxide. Other steps may be performed for the final cycle of the batch. In some embodiments, the final cycle of the batch includes another stripping step using about 20% ethanol of about two column volumes, another stripping step using about two column volumes of WFI, and a final equilibrium step performed as described above.

[0490] In some embodiments, the HIC resin life is between about 1 and about 100 cycles, between about 50 and about 90 cycles, less than about 100 cycles, about 10 cycles, about 20 cycles, about 40 cycles, about 50 cycles, about 60 cycles, about 70 cycles, about 80 cycles, about 90 cycles, or about 100 cycles. In some embodiments, the AEX resin life can reach 200 cycles.

[0491] Size Exclusion Chromatography

[0492] Size exclusion chromatography or gel filtration relies on the separation of components depending on their molecular size. The separation depends on the amount of time a substance spends in a porous stationary phase compared to the time in the fluid. The probability that a molecule will stay in a pore depends on the size of the molecule and the pore. In addition, the ability of a substance to penetrate into a pore is determined by the diffusion mobility of the macromolecule, which is higher for small macromolecules. Very large macromolecules simply cannot penetrate the pores of the stationary phase, and for very small macromolecules, the probability of penetration is close to unity. Components with larger molecular sizes move through the stationary phase more quickly, while components with small molecular sizes have a longer path length through the pores of the stationary phase and therefore stay in the stationary phase for a longer time.

[0493] The chromatographic material may comprise a size exclusion material, wherein the size exclusion material is a resin or a membrane. The matrix used for size exclusion is preferably an inert gel medium, which may be a complex of cross-linked polysaccharides, such as cross-linked agarose and / or dextran in the form of spherical beads. The degree of cross-linking determines the size of the pores present in the expanded gel beads. Molecules larger than a certain size will not enter the gel beads, and therefore move through the chromatographic bed as quickly as possible. Smaller molecules such as detergents, proteins, DNA, etc., which enter the gel beads to varying degrees depending on their size and shape, slow down to varying degrees in the passage through the bed. Therefore, molecules are generally eluted in the order of decreasing molecular size.

[0494] The porous chromatographic resin suitable for the size exclusion chromatography of virus can be made of dextrose, agarose, polyacrylamide or silicon dioxide with different physical characteristics.Polymer combinations can also be used.The most commonly used is the polymer combination of "SEPHADEX" that can be purchased from the trade name of Amersham Biosciences.Other size exclusion carriers from different construction materials are also suitable, such as Toyopearl 55F (polymethacrylate, from Tosoh Bioscience, Montgomery Pa., Montgomery, Pennsylvania) and Bio-Gel P-30Fine (BioRad Laboratories, Hercules, CA, Hercules, CA) of Hercules, California.

[0495] Virus Retention Filtration

[0496] Virus filtration is a dedicated virus reduction step in the production process. This step is usually performed after chromatographic polishing. Virus reduction by virus retention membranes is based on hydrodynamic radius differences, where proteins of interest, such as monoclonal antibodies (hydrodynamic radius of about 10 nm) pass through the filter, while larger viruses (hydrodynamic radius greater than 18 nm) are retained by the membrane.

[0497] Virus reduction can be achieved by using suitable filters, including (but not limited to) Planova 20N from Asahi Kasei Pharmaceuticals TM , 50N or BioEx; EMD Millipore's Viresolve TM Filter; Sartorius CPV or HF; or Pall's Ultipor DV20 filter, DV50 TM Or Pegasus Prime filter. Selecting an appropriate filter to obtain the desired filtering performance is obvious to those of ordinary skill in the art.

[0498] The virus retention filtration step may additionally include a pre-filtration step. Due to the pore blockage caused by impurities present in the feed stream, the virus retention filter is prone to premature fouling. Because the flux attenuation of the relevant virus retention filter is related to the viral breakthrough of some virus retention filters, fouling is undesirable. Studies have reported the use of a protective filter or a pre-filter to improve virus filter fouling. The inventors have found that chemically functionalized pre-filters (including HIC, CEX and AEX modes) can improve virus retention filtration capacity. Pre-filtration can be achieved by using a suitable pre-filter, the filter includes (but is not limited to) Viresolve Shield, Viresolve Shield H, Millistak + HC Pro XOSP (Millipore Sigma (MilliporeSigma)) or Pegasus Protect of Pall Corporation. It will be apparent to those of ordinary skill in the art that a suitable pre-filter is selected to obtain the desired filtration performance.

[0499] In some embodiments, a virus retaining filtration (VRF) step is performed immediately after the HIC step. In some embodiments, the load material is conditioned by three pre-filters immediately preceding the three filters.

[0500] In some exemplary embodiments, the VRF process includes the following six steps. The filter assembly is first primed and wetted with WFI until the device is filled and properly vented at an operating transmembrane pressure (TMP) of 5±2 psi. The second step includes venting with more than 50 L / m at an operating TMP of 25±5 psi. 2 The fourth step includes buffer flushing and flux verification: use more than 20L / m at 25±5psi TMP. 2 The assembly was equilibrated with a buffer comprising about 40 mM Tris and about 40 mM sodium citrate at pH 6.5 in a volume of 500 μL.

[0501] Then, at a constant TMP of 25 ± 5 psi, the flow rate was less than 900 L / m 2 The sample is loaded with a volume of about 700 L / m3, such as a HIC pool. Pool collection begins immediately after loading begins and ends once the loading material is exhausted. In some embodiments, the sample volume is about 700 L / m3. 2 About 1300L / m 2 Between, about 700L / m 2 , about 750L / m 2 , about 800L / m 2 , about 850L / m 2 , about 900L / m 2 , about 950L / m 2 , about 1000L / m 2 , about 1050L / m 2 , about 1100L / m 2 , about 1150L / m 2 , about 1200L / m 2 , about 1250L / m 2 , about 1300L / m 2 , about 1350L / m 2 , about 1400L / m 2 , about 1500L / m 2 , about 1600L / m 2 , about 1700L / m 2 , about 1800L / m 2 , about 1900L / m 2 or about 2000L / m 2In some embodiments, the loading conductivity is between about 7.5 mS / cm and about 13.5 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm, about 9.0 mS / cm, about 9.5 mS / cm, about 10.0 mS / cm, about 10.5 mS / cm, about 11.0 mS / cm, about 11.5 mS / cm, about 12.0 mS / cm, about 12.5 mS / cm, about 13.0 mS / cm, or about 13.5 mS / cm. In some embodiments, the loading pH is between about 4.5 and about 7.5, between about 6.3 and about 6.7, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5. In some embodiments, the loading protein concentration is between about 4.5 g / L and about 13 g / L, between about 8 g / L and about 10 g / L, about 4.5 g / L, about 5.0 g / L, about 5.5 g / L, about 6.0 g / L, about 6.5 g / L, about 7.0 g / L, about 7.5 g / L, about 8.0 g / L, about 8.5 g / L, about 9.0 g / L, about 9.5 g / L, about 10.0 g / L, about 10.5 g / L, about 11.0 g / L, about 11.5 g / L, about 12.0 g / L, about 12.5 g / L, or about 13.0 g / L.

[0502] Finally, the filter is flushed with WFI in preparation for final post-use integrity testing using sterile oil-free air or nitrogen.

[0503] Concentration and diafiltration

[0504] Certain exemplary embodiments of the present disclosure employ ultrafiltration and diafiltration (UF / DF), also referred to as concentration and diafiltration, to further concentrate and formulate the protein of interest. UF / DF regulates the API to achieve pH, excipient content, and protein concentration that are conducive to long-term storage and the addition of stabilizing excipients to produce the final API (FDS). During processing, the protein solution is pumped tangentially across the surface of a semipermeable parallel flat plate membrane. The membrane may have a pore size of, for example, about 50 kDa, which is permeable to water and buffer salts, but is generally impermeable to the protein of interest (e.g., a monoclonal antibody). The driving force for osmosis is the applied transmembrane pressure (TMP) caused by flow restriction at the outlet of the membrane flow channel.

[0505] Ultrafiltration is described in detail in Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, NY, 1996); and in Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9); the entire teachings of which are incorporated herein by reference. One filtration method is tangential flow filtration as described in the Millipore catalog entitled "Pharmaceutical Process Filtration Catalogue", pages 177-202 (Bedford, Mass., 1995 / 96), the entire teachings of which are incorporated herein by reference. Ultrafiltration is generally considered to mean filtration using filters with pore sizes less than 0.1 μm. By employing filters of such small pore size, the volume of the sample can be reduced by allowing the sample buffer to permeate the filter membrane pores, while the proteins are retained on the membrane surface.

[0506] UF / DF usually has three to four stages: initial concentration, diafiltration, secondary concentration and final concentration. During the initial concentration, TMP drives water and salts through the osmotic membrane, which reduces the liquid volume and thus increases the protein concentration. The degree of concentration of the initial concentration can be optimized to balance the output and protein stability.

[0507] During the diafiltration phase, permeable solutes are replaced as new buffer is flushed into the product stream. When new buffer is added at the same rate while removing permeate from the system, the sum of the retentate tank and the slide volume defines the system volume. A flow-through volume (turn-over volume; TOV) is defined as the amount of diafiltration buffer added to the UF / DF process and matching the system volume. Typically, replacing 8 system volumes (8TOV) ensures >99.9% buffer exchange. The diafiltration buffer is intended to adjust the protein to a stable pH and excipient concentration that compensates for high product concentrations.

[0508] Once sufficient buffer exchange has occurred, a secondary concentration further reduces the product volume to facilitate storage and formulation. If further reduction of the product volume is required, for example due to equipment assembly considerations, higher final protein concentration requirements, or desired adjustment of viscosity modifiers, a final concentration is performed.

[0509] A person skilled in the art can select an appropriate membrane filtration device for UF / DF operation. Examples of membrane cassettes suitable for the present invention include, but are not limited to, Pellicon 2 or Pellicon 3 cassettes containing 10 kD, 30 kD or 50 kD membranes from EMD Millipore; Kvick 10 kD, 30 kD or 50 kD membrane cassettes from Stovam; and Centramate or Centrasette 10 kD, 30 kD or 50 kD cassettes from Pall Corporation.

[0510] The exemplary steps are described again in more detail. The first UF / DF step includes a WFI flush with a retentate volume of approximately 12 L / m 2 And the permeate is 40L / m 2 The feed flow rate is between about 200 and 300 L / hr / m 2 (LMH) at a pressure of about 25 ± 5 psi. Next, a pre-use leak test was performed with WFI at an inlet pressure of 8-12 psi. This was followed by equilibration with about 4 mM acetate at a pH of about 4.10 ± 0.10. The volume of the equilibration buffer was greater than or about 5 L / m 2 , and the flow rate is between about 200 and 300LMH.

[0511] The sample to be concentrated (loaded) is then adjusted for compatibility with the final drug substance composition. The loading adjustment solution can be, for example, about 10% (w / v) ultrafine polysorbate 80 or polysorbate 20, added at about 50 μL / L of load.

[0512] Next, an initial concentration step is performed with the load, such as the VRF pool. 2 The volume of the mixture is added at a flow rate of about 200 to about 350 LMH, and a pressure of about 20 ± 10 psi. In an exemplary embodiment, the flow rate is about 300 LMH. The protein concentration after initial concentration can be between about 60 and 80 g / L. In an exemplary embodiment, the protein concentration of this step is about 70 g / L.

[0513] The initial concentration step is followed by a diafiltration step. The diafiltration buffer may comprise about 4 mM acetate at a pH between about 4.00 and 4.20. In some embodiments, the concentration of acetate in the diafiltration buffer is between about 3 mM and about 10 mM, between about 3 mM and about 5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM. In some embodiments, the pH of the diafiltration buffer is between about 4.00 and about 4.50, about 4.00, about 4.05, about 4.10, about 4.15, about 4.20, about 4.25, about 4.30, about 4.35, about 4.40, about 4.45, or about 4.50. In an exemplary embodiment, the pH of the diafiltration buffer is about 4.10.

[0514] The volume of the diafiltration buffer used can be about or greater than 8TOV, the flow velocity is between about 200 and 350LMH, and the pressure is between about 10 and 30psi. In an exemplary embodiment, the flow velocity of the diafiltration is about 300LMH. In an exemplary embodiment, the pressure of the diafiltration is about 20psi. The protein concentration after the diafiltration can be between about 60 and about 80g / L. In an exemplary embodiment, the protein concentration after the diafiltration is about 70g / L.

[0515] The diafiltration is followed by a secondary concentration step. The amount of product used for the secondary concentration is about or less than 970 g / m 2 The flow rate of the secondary concentration is between about 100 and about 350 LMH. In an exemplary embodiment, the flow rate is about 300 LMH. The pressure of the secondary concentration is between about 10 and 30 psi, and the inlet pressure is less than 60 psi. The protein concentration after the secondary concentration can be between about 80 and 100 g / L. In an exemplary embodiment, the protein concentration after the secondary concentration is about 90 g / L.

[0516] The secondary concentration is followed by a membrane depolarization step. The duration of this process can be between about 5 and 15 minutes, with a flow rate of about or less than 350 LMH and a pressure of less than 30 psi. For this step, the permeate valve is opened 0% and the retentate valve is opened 100%.

[0517] This is followed by a final or third concentration step. The amount of final concentration load used may be about or less than 1800 g / m 2, with a flow rate between about 5 and 350LMH and an inlet pressure less than 60psi added. In some embodiments, the final protein concentration is between about 180g / L and about 260g / L, between about 225g / L and about 255g / L, between about 228g / L and about 232g / L, for about 180g / L, about 185g / L, about 190g / L, about 195g / L, about 200g / L, about 205g / L, about 210g / L, about 215g / L, about 220g / L, about 225g / L, about 230g / L, about 235g / L, about 240g / L, about 245g / L, about 250g / L, about 255g / L or about 260g / L. In an exemplary embodiment, the final protein concentration is about 232g / L. In another exemplary embodiment, the final protein concentration is about 240 g / L.

[0518] After final concentration, the method includes another membrane depolarization step for about 15 to 35 minutes. In an exemplary embodiment, the duration of this step is about 25 minutes. The feed flow rate is about or less than 350LMH, the pressure is less than 30psi, the permeate valve is opened 0%, and the retentate valve is opened 100%.

[0519] Finally, after membrane polarization, there is a WFI flush step, applying 10 L / m at a flow rate between about 200 and about 250 LMH. 2 WFI.

[0520] preparation

[0521] Certain exemplary embodiments further comprise conditioning the final concentrated pool (FCP) protein after the concentration and diafiltration steps. The concentrated protein can be adjusted to a desired concentration with desired excipients for use in the final drug substance (FDS).

[0522] If the filtered FCP protein concentration is above the specified target, it is diluted with FCP dilution buffer to adjust it to the target concentration. The FCP dilution buffer may comprise about 10 mM sodium acetate, about 25 mM arginine hydrochloride, and about 5% sucrose, with a pH of about 5.3. In some embodiments, the concentration of sodium acetate in the FCP dilution buffer may be between about 2.0 mM and about 14.0 mM, between about 6.0 mM and about 12.0 mM, between about 8.0 mM and about 10.0 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6.0 mM, about 7.0 mM, about 8.0 mM, about 9.0 mM, about 10.0 mM, about 11.0 mM, about 12.0 mM, about 13.0 mM, about 14.0 mM, about 15.0 mM, about 16.0 mM, about 17.0 mM, about 18.0 mM, about 19.0 mM, about 20.0 mM, about 21.0 mM, about 23.0 mM, about 24.0 mM, about 25.0 mM, about 26.0 mM, about 27.0 mM, about 28.0 mM, about 29.0 mM, about 30.0 mM, about 31.0 mM, about 32.0 mM, about 33.0 mM, about 34.0 mM, about 35.0 mM, about 36.0 mM, about 37.0 mM, about 38.0 mM, about 39.0 mM, In some embodiments, the concentration of arginine hydrochloride in the FCP dilution buffer can be between about 10 mM and about 40 mM, between about 15 mM and about 35 mM, between about 20 mM and about 30 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, or about 40 mM. In some embodiments, the concentration of sucrose in the FCP dilution buffer can be between about 1% and 10%, between about 2% and 9%, between about 3% and 8%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the pH of the FCP dilution buffer can be between about 4.0 and about 6.5, between about 4.5 and about 6.0, between about 5.0 and about 5.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, or about 7.0.

[0523] In some embodiments, the FCP can be further combined with an excipient concentration buffer to achieve both a target dilution of the FCP protein and a target excipient concentration. The FCP excipient concentration buffer can comprise about 10 mM acetate, about 350 mM arginine hydrochloride, and about 70% (w / v) sucrose, with a pH of about 5.3. In some embodiments, the concentration of acetate in the excipient concentration buffer can be between about 2.0 mM and about 14.0 mM, between about 6.0 mM and about 12.0 mM, between about 8.0 mM and about 10.0 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6.0 mM, about 7.0 mM, about 8.0 mM, about 9.0 mM, about 10.0 mM, about 11.0 mM, about 12.0 mM, about 13.0 mM, about 14.0 mM, about 15.0 mM, about 16.0 mM, about 17.0 mM, about 18.0 mM, about 19.0 mM, about 20.0 mM, about 21.0 mM, about 23.0 mM, about 24.0 mM, about 25.0 mM, about 26.0 mM, about 27.0 mM, about 28.0 mM, about 29.0 mM, about 30.0 mM, about 31.0 mM, about 32.0 mM, about 33.0 mM, about 34.0 mM, about 35.0 mM, about 36.0 mM, about 37.0 mM, about 38. In some embodiments, the concentration of arginine hydrochloride in the excipient concentration buffer may be between about 200mM and about 500mM, between about 250mM and about 450mM, between about 300mM and about 400mM, about 200mM, about 250mM, about 300mM, about 350mM, about 400mM, about 450mM, or about 500mM. In some embodiments, the concentration of sucrose in the excipient concentration buffer may be between 50% and 90%, between about 55% and 85%, between about 60% and 80%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, or about 85%. In some embodiments, the pH of the excipient concentration buffer may be between about 4.0 and about 6.5, between about 4.5 and about 6.0, between about 5.0 and about 5.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, or about 7.0.

[0524] After dilution with dilution buffer and combination with excipient concentration buffer, the conditioned FCP can be filtered into a disposable bag and mixed. The sample can be referred to as filtered drug substance (DS). The filtered DS can be further diluted to the target concentration with FDS dilution buffer and mixed with excipient buffer solution to the final concentration and composition.

[0525] The target concentration may be about 200.0 mg / mL. In some embodiments, the target concentration is between about 160.0 mg / mL and about 240.0 mg / mL, between about 180.0 mg / mL and about 220.0 mg / mL, between about 190.0 mg / mL and about 210.0 mg / mL, between about 195.0 mg / mL and about 205.0 mg / mL, between about 199.0 mg / mL and about 201.0 mg / mL, between about 199.5 mg / mL and about 200.5 mg / mL, between about 199.9 mg / mL and about 200.1 mg / mL, about 160.0 mg / mL, about 165.0 mg / mL, about 170.0 mg / mL, about 180.0 mg / mL, about 191.0 mg / mL, about 181.0 mg / mL, about 183.0 mg / mL, about 187.0 mg / mL, about 193.0 mg / mL, about 194.0 mg / mL, about 195.0 mg / mL, about 205.0 mg / mL, about 196.0 mg / mL, about 207.0 mg / mL, about 213.0 mg / mL, about 214.0 mg / mL, about 215.0 mg / mL, about 220.0 mg / mL, about 221.0 mg / mL, about 223.0 mg / mL, about 224.0 mg / mL, about 225.0 mg / mL, about 226.0 mg / mL, about 227.0 mg / mL, about 228.0 mg / mL, about 229.0 mg / mL, about 234.0 mg / mL, about 236.0 mg / mL, about 237.0 mg / mL, about 238.0 about 175.0 mg / mL, about 180.0 mg / mL, about 185.0 mg / mL, about 190.0 mg / mL, about 195.0 mg / mL, about 199.0 mg / mL, about 199.5 mg / mL, about 199.9 mg / mL, about 200.0 mg / mL, about 200.1 mg / mL, about 200.5 mg / mL, about 201.0 mg / mL, about 205.0 mg / mL, about 210.0 mg / mL, about 215.0 mg / mL, about 220.0 mg / mL, about 225.0 mg / mL, about 230.0 mg / mL, about 235.0 mg / mL or about 240.0 mg / mL.

[0526] The FDS dilution buffer may comprise about 10 mM sodium acetate, about 25 mM arginine hydrochloride, and about 5% sucrose at a pH of about 5.3. In some embodiments, the concentration of sodium acetate in the FDS dilution buffer may be between about 2.0 mM and about 14.0 mM, between about 6.0 mM and about 12.0 mM, between about 8.0 mM and about 10.0 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6.0 mM, about 7.0 mM, about 8.0 mM, about 10.0 mM, about 12 ... In some embodiments, the concentration of arginine hydrochloride in the FDS dilution buffer may be between about 10 mM and about 40 mM, between about 15 mM and about 35 mM, between about 20 mM and about 30 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, or about 40 mM. In some embodiments, the concentration of sucrose in the FDS dilution buffer may be between about 1% and 10%, between about 2% and 9%, between about 3% and 8%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the pH of the FDS dilution buffer may be between about 4.0 and about 6.5, between about 4.5 and about 6.0, between about 5.0 and about 5.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, or about 7.0.

[0527] The composition of the excipient buffer solution can be selected depending on the target concentration and composition of the final drug substance (FDS).

[0528] The excipient buffer solution with a target FDS concentration of 150 mg / mL may include about 20 mM sodium acetate, about 80 mM L-histidine, about 25 mM L-arginine hydrochloride, about 5% sucrose, and about 0.8% polysorbate 80, with a pH of about 6.7. In some embodiments, the concentration of sodium acetate in the FDS excipient buffer solution (target FDS concentration of 150 mg / mL) may be between about 5 mM and about 35 mM, between about 10 mM and about 30 mM, between about 15 mM and about 25 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, or about 35 mM. In some embodiments, the concentration of L-histidine in the FDS excipient buffer solution (target FDS concentration is 150 mg / mL) can be between about 60 mM and about 100 mM, between about 65 mM and about 95 mM, between about 70 mM and about 90 mM, between about 75 mM and about 85 mM, about 60 mM, about 70 mM, about 80 mM or about 90 mM. In some embodiments, the concentration of L-arginine hydrochloride in the FDS excipient buffer solution (target FDS concentration is 150 mg / mL) can be between about 10 mM and about 40 mM, between about 15 mM and about 35 mM, between about 20 mM and about 30 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM or about 40 mM. In some embodiments, the concentration of sucrose in the FDS excipient buffer solution (target FDS concentration of 150 mg / mL) may be between about 1% and 10%, between about 2% and 9%, between about 3% and 8%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the concentration of polysorbate 80 in the FDS excipient buffer solution (target FDS concentration of 150 mg / mL) may be between about 0.1% and 1.6%, between about 0.3% and 1.3%, between about 0.6% and 1.1%, between about 0.8% and 0.9%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1.0%, about 1.2%, about 1.4%, or about 1.6%. In some embodiments, the pH of the FDS excipient buffer solution (target FDS concentration of 150 mg / mL) can be between about 5.0 and about 8.0, between about 5.5 and about 7.5, between about 6.0 and about 7.0, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5.

[0529] The excipient buffer solution with a target FDS concentration of 175 mg / mL may include about 30 mM sodium acetate, about 160 mM L-histidine, about 225 mM L-arginine hydrochloride, about 5% (w / v) sucrose, and about 1.6% (w / v) polysorbate 80, with a pH of about 7.0. In some embodiments, the concentration of sodium acetate in the FDS excipient buffer solution (target FDS concentration of 175 mg / mL) may be between about 15 mM and about 45 mM, between about 20 mM and about 40 mM, between about 25 mM and about 35 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 45 mM. In some embodiments, the concentration of L-histidine in the FDS excipient buffer solution (target FDS concentration of 175 mg / mL) can be between about 120 mM and about 200 mM, between about 130 mM and about 190 mM, between about 140 mM and about 180 mM, between about 150 mM and about 170 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, or about 180 mM. In some embodiments, the concentration of L-arginine hydrochloride in the FDS excipient buffer solution (target FDS concentration is 175 mg / mL) can be between about 125 mM and about 325 mM, between about 150 mM and about 300 mM, between about 175 mM and about 275 mM, between about 200 mM and about 250 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 275 mM, about 300 mM or about 325 mM. In some embodiments, the concentration of sucrose in the FDS excipient buffer solution (target FDS concentration is 175 mg / mL) can be between about 1% and 10%, between about 2% and 9%, between about 3% and 8%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10%. In some embodiments, the concentration of polysorbate 80 in the FDS excipient buffer solution (target FDS concentration of 175 mg / mL) can be between about 0.8% and 2.4%, between about 1.0% and 2.2%, between about 1.2% and 2.0%, between about 1.4% and 1.8%, about 0.8%, about 1.2%, about 1.4%, about 1.6%, about 1.8%, about 2.0%, about 2.2%, or about 2.4%. In some embodiments, the pH of the FDS excipient buffer solution (target FDS concentration of 175 mg / mL) can be between about 5.5 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0.

[0530] Example

[0531] Example 1. Insulin supplementation

[0532] Total insulin concentrations (0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L) and insulin schedules (add all on day 4; add equally on days 2 and 4; add equally on days 2, 4, 6, and 8) were assessed in combination.

[0533] The custom D-optimal design was inoculated as shown in the following table (Table 9) using modified seed expansion cultures in flasks and bioreactors using normal operating conditions. A 15-day fed-batch process was performed in a 2L bioreactor with CDM1B containing taurine and sodium phosphate (Table 10).

[0534] Table 9 (Insulin supplementation experimental conditions)

[0535]

[0536] Table 10 (Insulin supplementation 2L bioreactor conditions)

[0537]

[0538] Cell counts and metabolites of bioreactor samples were measured on a Nova Flex analyzer. After production was completed, samples were analyzed for titer analysis. When production duration was 15 days, day 13 bioreactor samples were analyzed for product quality analysis.

[0539] In some bioreactors, increased insulin resulted in higher productivity ( Figure 2 ), higher survival rate ( Figure 3 ) and overall reduction in ammonia ( Figure 4 ). Higher inoculation density also affected titer curve parameters and caused a decrease in ammonia and survival rate.

[0540] According to JMP optimization ( Figure 5 ), it was decided to increase the insulin concentration to 15 mg / L. For maximum titer, providing insulin on days 2 and 4 is preferred. Therefore, it was found that an insulin feeding strategy of 7.5 mg / L on days 2 and 4 was optimal.

[0541] Product quality was monitored regularly throughout the culture and feed incubation period. Bioreactor samples were collected on day 13 for product quality analysis. Galactosylation and fucosylation were not affected by process variables. The effect of insulin feed schedule on aggregates ( Figure 6 ), non-reducing purity ( Figure 7 ) and NGHC( Figure 8 The impact of insulin feeding schedule on these product quality attributes was minor and within the cultivation targets.

[0542] Example 2. Tyrosine supplementation

[0543] It should be noted that during the increased productivity several amino acids were depleted. Therefore, additional tyrosine feed was investigated to address the depletion issue.

[0544] The modified seed expansion culture performed in the flask using normal operating conditions was used, and the flasks were inoculated with a custom D-optimal design as shown in the following table (Table 11). A 15-day batch feed process was performed in a 250 mL flask with a cell culture medium containing sodium phosphate and taurine. Tyrosine was supplemented on the 3rd day of production. According to the experimental design, tyrosine concentration varied between 1.8 g / L and 2.2 g / L (Table 11).

[0545] Table 11

[0546]

[0547] Feeding additional tyrosine into the cell production causes ammonia reduction and has minimal effect on productivity, e.g. Fig. 9 as shown in .

[0548] Example 3. Phosphate redistribution

[0549] Phosphate is essential for energy transfer in cells and for the synthesis of nucleic acids and phospholipids. Phosphate depletion can lead to reduced cell viability and accumulation of undesirable cell metabolism byproducts. Initial small-scale model development experiments indicated that phosphate may be depleted on day 2. Therefore, phosphate redistribution was studied, including an increase in basal medium concentration.

[0550] Evaluating phosphate feeding schedules in production flask models. Avoiding depletion of key nutrients, including phosphate, is essential and necessary before initiating more complex media and feeding studies.

[0551] Use the seed expansion culture through modification, inoculate the production flask experiment utilizing flask and seed expansion culture bioreactor.In the flask with the initial production medium that comprises taurine, carry out the batch feed process of selected cell line.According to the experimental design, the sodium phosphate content in the initial medium changes.In whole production, the sodium phosphate in four feedings is also different (each condition n=2, referring to Table 12).

[0552] Table 12

[0553]

[0554]

[0555] * Component content is defined as the concentration before inoculation

[0556] Daily samples were analyzed for cell counts on a Nova CDV, metabolites on a Nova BP400, and osmolality on an Advanced Instruments Model 2020 osmometer. Samples were also provided for titer analysis.

[0557] The phosphate redistribution strategy resulted in a slight increase in final titer ( Fig.10 Different feed patterns changed the shape of the phosphate concentration curve, and three conditions approached depletion at different production days ( Fig.11 The conditions where phosphate was increased early in the batch and continued to be fed until the end of the batch (267, 525, 525, 250, 250 mg / L) did not deplete phosphate during production; this feeding strategy was implemented for the upstream process.

[0558] Example 4. Production bioreactor conditions

[0559] Cell culture parameters in the production bioreactor are expected to have the greatest impact on product quality and process productivity because the majority of protein is produced in this unit operation.

[0560] Two N-3 10L stirred tank bioreactors were inoculated with the culture from the seed expansion. After reaching the N-3 final density, three N-2 10L stirred tank bioreactors were inoculated. After reaching the N-2 final density, four N-1 10L stirred tank bioreactors were inoculated. Appropriate split ratios were used throughout the seed expansion culture amplification. Under the three specified final N-1 density targets, 2L batch feed production bioreactors were inoculated. Three of the four N-1 reactors were used to inoculate the production bioreactor. Except for the inoculation-related variables that set the density, temperature and pH upper limits according to the experimental design, the pH lower limit remained constant.

[0561] Daily samples were analyzed for cell counts and metabolites on the Nova FLEX. After the production batch was completed, day 10, day 12, and / or day 13 samples were analyzed for titer analysis and quality testing. Sixteen samples from day 10, day 12, and / or day 13 were also analyzed for sequence variants.

[0562] It was determined that the collection day had a large impact on initial general quality attributes, process attributes, and sequence variants (titer, total fucosylation, total galactosylation, viability, total Pro-Ala SV, Pro-Ala site P12). The collection day was a time-dependent upstream process parameter where the operating range was defined taking into account production equipment capacity and flexibility. Monte Carlo simulations were performed to determine the combination of ranges. The best profiler based on the JMP desirability maximization algorithm determined day 10.0 as the optimal set point.

[0563] Monte Carlo simulations were performed on the N-1 density to characterize the failure rate of the process and the quality attributes of the process range tested in combination. The ideal process range was then selected based on achieving the most desirable manufacturing process, with an emphasis on minimizing the failure rate (target failure rate <100ppm, 0.01%). Failures occurred primarily in meeting the Cys-Tyr substitution, SDS LMW, SDS purity, SDS purity (HC+LC+NGHC), SEC HMW dimer aggregates, and SEC HMW higher order aggregates targets. Increasing the upper limit of the N-1 density had minimal impact on the quality attributes, with a greater impact on Cys-Tyr substitution (2.0% increase). The N-1 density range was set to 4.35 to 5.45×10 5 cells / mL to balance quality attribute responses with manufacturing flexibility.

[0564] Increasing the upper limit of the collection day increases NR SDS LMW failure by 70% and reduces SEC HMW high-order aggregates by 22%. The increase of NR SDS LMW reduces SDS LMW and collection duration simultaneously due to the reduction of trisulfide. Due to this difference and in order to improve manufacturing simplicity, the collection day range is set to 10 to 11 days. For the final process, the final collection day range of 5 standard deviations and the N-1 density range are selected to balance stricter control (lower failure rate) and manufacturing flexibility. An overview of the simulation conditions can be found in Table 13 (Overview of Production Simulation Conditions).

[0565] Table 13 (Overview of production simulation conditions)

[0566]

[0567] Collection Overview

[0568] The collection process can begin at the separation step after the recombinant protein has been produced using the upstream production methods described above and / or by alternative production methods conventional in the art. When using the cell culture techniques of the present disclosure, the protein of interest can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. In embodiments in which the protein of interest is produced intracellularly, host cells or lysed cell particulate debris (e.g., resulting from homogenization) can be removed by various means, including (but not limited to) centrifugation or ultrafiltration. In the case where the protein of interest is secreted into the culture medium, a commercially available protein concentration filter can be first used, such as using Amicon TM or Millipore Pellicon TM Ultrafiltration units are used to concentrate supernatants from such expression systems. In one aspect, the protein of interest can be collected by centrifugation, followed by depth filtration and subsequent affinity capture chromatography.

[0569] It is contemplated that a variety of different production techniques are within the scope of the present invention, including, but not limited to, affinity chromatography, ion exchange chromatography, mixed mode chromatography, size exclusion chromatography, and hydrophobic interaction chromatography, either alone or in combination. These chromatographic steps separate a mixture of proteins of a biological sample based on their charge, hydrophobicity, or size, or a combination thereof, depending on the particular separation format. Several different chromatographic resins can be used for each of the aforementioned techniques, allowing the production process to be precisely modified for the specific protein involved. Each separation method allows the protein to pass through the column at a different rate to achieve physical separation, which increases as the protein passes further through the column or selectively attaches to the separation medium. The protein is then (i) eluted differently using an appropriate elution buffer, and / or (ii) collected by a flow-through fraction obtained from the column used, optionally by washing the column with an appropriate equilibration buffer. In some cases, when impurities preferentially attach to the column and the protein of interest is less attached to the column (e.g., the protein of interest is not adsorbed to the solid phase of a particular column and therefore flows through the column), the protein of interest is separated from the impurities (HCP, protein variants, etc.). In some cases, impurities are separated from the protein of interest when they fail to adsorb to the column and therefore flow through the column.

[0570] The collection process may start at a separation step after the recombinant protein has been produced using the upstream production methods described above and / or by conventional alternative production methods in the art. After a clear solution or mixture containing the protein of interest (e.g., dupilumab) has been obtained, the protein of interest is separated from process-related impurities (e.g., other proteins produced by the cell (e.g., HCPs) and product-related substances, such as acidic or basic variants). A combination of one or more different production techniques may be used, including affinity chromatography, ion exchange (e.g., CEX, AEX) chromatography, mixed mode (MM) chromatography, and / or hydrophobic interaction chromatography. Such production steps separate mixtures of components based on, for example, the charge, hydrophobicity, and / or apparent size of the components within a biological sample. A variety of chromatographic resins are commercially available for use in each of the chromatographic techniques mentioned herein, so that the production process is precisely modified for the specific protein involved. Each separation method allows the protein to pass through the column at different rates to achieve physical separation, which increases as the protein passes further through the column; or allows the protein to be selectively adsorbed to the separation resin (or culture medium). The protein may then be collected differently. In some cases, the protein of interest is separated from components of the biological sample when other components are specifically adsorbed to the resin of the column but the protein of interest is not adsorbed to the resin.

[0571] The column loading and washing steps can be controlled by online, at-line or offline measurements of the content of product-related impurities / substances in the column effluent or the collected pool or both, so as to achieve a specific target product quality and / or yield. In certain embodiments, the loading concentration can be dynamically controlled by in-line or batch or serial dilution with a buffer or other solution...

Claims

1. A system for producing dupilumab, comprising: (g) a bioreactor for culturing cells capable of expressing dupilumab; (h) one or more stirring elements, wherein the one or more stirring elements are configured below an initial working volume of the bioreactor; and (i) one or more gas control components coupled to the bioreactor for controlling dissolved gases.

2. The system of claim 1, wherein the bioreactor volume is greater than or equal to 500 L.

3. The system of claim 1, wherein the bioreactor volume is greater than or equal to 3,000 L.

4. The system of claim 1, wherein the bioreactor volume is greater than or equal to 10,000 L.

5. The system of claim 1, wherein the one or more agitation elements comprise one or more impeller assemblies.

6. The system of claim 5, wherein the one or more stirring elements are configured to have an initial stirring rate between 20 rpm and 150 rpm.

7. The system of claim 6, wherein the one or more stirring elements are configured to have a stirring rate that can increase by 25%, 50%, 75%, 100%, 125%, 150%, 175% or 200% on one or more days selected from the following: day 0.5, day 1, day 1.5, day 2, day 2.5, day 3, day 3.5, day 4, day 4.5, day 5, day 5.5, day 6, day 6.5, day 7, day 7.5, day 8, day 8.5, day 9, day 9.5, day 10, day 10.5 and day 11.

8. The system of claim 1, wherein the one or more gas control components include one or more bubblers.

9. The system of claim 8, wherein the one or more bubblers are configured to have an initial bubbling rate of about 25 to 75 slpm.

10. The system of claim 9, wherein the one or more bubblers are configured to have a bubbling rate that can increase by 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% on one or more days selected from the group consisting of: day 0.5, day 1, day 1.5, day 2, day 2.5, day 3, day 3.5, day 4, day 4.5, day 5, day 5.5, day 6, day 6.5, day 7, day 7.5, day 8, day 8.5, day 9, day 9.5, day 10, day 10.5, and day 11.

11. The system of claim 8, wherein the one or more bubblers are configured to automatically adjust a bubbling rate based on dissolved oxygen levels.

12. The system of claim 9, wherein the one or more bubblers include 146 to 292 holes having a size between 0.5 mm and 2 mm.

13. A method for enhancing cell growth, cell viability, cell density or dupilumab production in a mammalian cell culture process, comprising the steps of: (d) varying the agitation rate at different time points during the growth and production phases; (e) varying the sparging rate at different time points during the growth and production phases; and (f) Changing the target level of dextrose at different time points during the growth and production phases.

14. The method of claim 13, wherein the initial stirring rate is set between 20 rpm and 150 rpm.

15. The method of claim 13, wherein the stirring rate is configured to increase by 25%, 50%, 75%, 100%, 125%, 150%, 175% or 200% on one or more days selected from the group consisting of: day 0.5, day 1, day 1.5, day 2, day 2.5, day 3, day 3.5, day 4, day 4.5, day 5, day 5.5, day 6, day 6.5, day 7, day 7.5, day 8, day 8.5, day 9, day 9.5, day 10, day 10.5 and day 11.

16. The method of claim 13, wherein more than one bubbler is used to vary the bubbling rate.

17. The method of claim 16, wherein the bubbler is set to an initial bubbling rate of about 25 to 75 slpm.

18. The method of claim 17, wherein the bubbling rate is increased by 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% on one or more days selected from the group consisting of: day 0.5, day 1, day 1.5, day 2, day 2.5, day 3, day 3.5, day 4, day 4.5, day 5, day 5.5, day 6, day 6.5, day 7, day 7.5, day 8, day 8.5, day 9, day 9.5, day 10, day 10.5, and day 11.

19. The method of claim 13, wherein the sparging rate is automatically adjusted based on dissolved oxygen levels.

20. The method of claim 16, wherein the bubbler comprises 146 to 292 holes having a size between 0.5 mm and 2 mm.

21. The method of claim 13, wherein the target level of initial dextrose is set between 5 g / L and 7 g / L.

22. The method of claim 13, wherein the target level of dextrose is set to vary between 5 g / L and 7 g / L on day 0, and then incremented to vary between 7 g / L and 9 g / L on day 2, and then incremented to vary between 9 g / L and 11 g / L on day 4.

23. The method of claim 13, wherein the target level of dextrose is set to vary between 5 g / L and 7 g / L on day 0, and then increased incrementally to vary between 7 g / L and 11 g / L on day 2, and then decreased to vary between 5 g / L and 7 g / L on day 4.

24. A method for producing an anti-IL-4Rα antibody or an antigen-binding fragment thereof, comprising the following steps: (a) culturing cells expressing an anti-IL-4Rα antibody or an antigen-binding fragment thereof in a cell culture medium, wherein the cumulative concentration of one or more polyamines in the cell culture medium is between about 0.03 and about 0.9 mM; (b) agitating the cell culture; and (c) controlling the dissolved gas concentration in the cell culture.

25. The method of claim 24, wherein the cell culture medium is subjected to a high temperature short time (HTST) treatment of about 101°C to 106°C for 8 to 15 seconds.

26. The method of claim 24, wherein the two or more impeller assemblies are positioned below the surface of the initial working volume.

27. The method of claim 24, wherein agitation of the cell culture is performed using one or more impeller assemblies, and an uppermost impeller is positioned below the surface of the initial working volume.

28. The method of claim 24, wherein the initial stirring rate is configured to be between 20 rpm and 150 rpm.

29. The method of claim 24, wherein the stirring rate is configured to increase by 25%, 50%, 75%, 100%, 125%, 150%, 175% or 200% on one or more days selected from the group consisting of: day 0.5, day 1, day 1.5, day 2, day 2.5, day 3, day 3.5, day 4, day 4.5, day 5, day 5.5, day 6, day 6.5, day 7, day 7.5, day 8, day 8.5, day 9, day 9.5, day 10, day 10.5 and day 11.

30. The method of claim 24, wherein the dissolved gas concentration is controlled by one or more bubblers.

31. The method of claim 30, wherein the one or more bubblers are configured for an initial bubbling rate of about 25 to 75 slpm.

32. The method of claim 31 , wherein the bubbling rate is configured to increase by 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% on one or more days selected from the group consisting of: day 0.5, day 1, day 1.5, day 2, day 2.5, day 3, day 3.5, day 4, day 4.5, day 5, day 5.5, day 6, day 6.5, day 7, day 7.5, day 8, day 8.5, day 9, day 9.5, day 10, day 10.5, and day 11.

33. The method of claim 30, wherein the sparging rate is automatically configured based on dissolved oxygen levels.

34. A bioreactor comprising: One or more optical probes located in a reservoir of the bioreactor for generating a data signal having reduced signal noise compared to electrochemical probes.

35. The bioreactor of claim 34, comprising two or more optical probes.

36. A bioreactor according to claim 35, wherein there are two or more optical probes arranged in the lower third of the reservoir of the bioreactor.

37. The bioreactor of claim 35 having two or more optical probes arranged at two different positions along the probe belt.

38. The bioreactor of claim 34, further comprising a stirring element comprising one or more impeller assemblies, wherein an uppermost impeller is positioned below the surface of the initial working volume.

39. A method for treating a patient suffering from a type 2 inflammatory disease, comprising administering to the patient dupilumab produced by the system of any one of claims 1 to 12.

40. The method of claim 39, wherein the type 2 inflammatory disease is atopic dermatitis, moderate to severe atopic dermatitis, asthma, moderate to severe asthma, allergic rhinitis, chronic sinusitis with nasal polyps, eosinophilic esophagitis, chronic obstructive pulmonary disease, chronic spontaneous urticaria, prurigo nodularis, allergic fungal sinusitis, chronic sinusitis without nasal polyps, allergies, grass allergies, peanut allergies, dairy allergies, bullous pemphigoid, atopic dermatitis of the hands and feet, cold urticaria, chronic induced urticaria, ulcerative colitis, chronic pruritus of unknown origin, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, bronchiectasis, or alopecia areata.

41. A method for treating a disease or condition associated with IL-4R activity, comprising administering to a patient dupilumab produced by the system of any one of claims 1 to 12.

42. The method of claim 41, wherein the disease or condition associated with IL-4R activity is atopic dermatitis, moderate to severe atopic dermatitis, asthma, moderate to severe asthma, allergic rhinitis, chronic sinusitis with nasal polyps, eosinophilic esophagitis, chronic obstructive pulmonary disease, chronic spontaneous urticaria, prurigo nodularis, allergic fungal sinusitis, chronic sinusitis without nasal polyps, allergies, grass allergies, peanut allergies, dairy allergies, bullous pemphigoid, atopic dermatitis of the hands and feet, cold urticaria, chronic induced urticaria, ulcerative colitis, chronic pruritus of unknown origin, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, bronchiectasis, or alopecia areata.

43. A method for treating a patient suffering from a type 2 inflammatory disease, comprising administering to the patient dupilumab produced according to the method of any one of claims 13 to 33.

44. The method of claim 39, wherein the type 2 inflammatory disease is atopic dermatitis, moderate to severe atopic dermatitis, asthma, moderate to severe asthma, allergic rhinitis, chronic sinusitis with nasal polyps, eosinophilic esophagitis, chronic obstructive pulmonary disease, chronic spontaneous urticaria, prurigo nodularis, allergic fungal sinusitis, chronic sinusitis without nasal polyps, allergies, grass allergies, peanut allergies, dairy allergies, bullous pemphigoid, atopic dermatitis of the hands and feet, cold urticaria, chronic induced urticaria, ulcerative colitis, chronic pruritus of unknown origin, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, bronchiectasis, or alopecia areata.

45. A method for treating a disease or condition associated with IL-4R activity, comprising administering to a patient dupilumab produced according to the method of any one of claims 13 to 33.

46. ​​The method of claim 41, wherein the disease or condition associated with IL-4R activity is atopic dermatitis, moderate to severe atopic dermatitis, asthma, moderate to severe asthma, allergic rhinitis, chronic sinusitis with nasal polyps, eosinophilic esophagitis, chronic obstructive pulmonary disease, chronic spontaneous urticaria, prurigo nodularis, allergic fungal sinusitis, chronic sinusitis without nasal polyps, allergies, grass allergies, peanut allergies, dairy allergies, bullous pemphigoid, atopic dermatitis of the hands and feet, cold urticaria, chronic induced urticaria, ulcerative colitis, chronic pruritus of unknown origin, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, bronchiectasis, or alopecia areata.

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