Cell culture methods for antibody production

Abstract

The present invention relates to methods for producing high titer antibody products. In particular, the present invention relates, in part, to improved serum-free animal cell culture media that can be used for the production of proteins of interest. Additionally, the present invention further relates to chromatographic methods used to successfully isolate the antibody product objects of the present disclosure.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application incorporates by reference and claims priority to and benefit of Provisional Patent Application No. 63 / 315,897, filed March 2, 2022; Provisional Patent Application No. 63 / 411,899, filed September 30, 2022; Provisional Patent Application No. 63 / 417,873, filed October 20, 2022; Provisional Patent Application No. 63 / 436,854, filed January 3, 2023, and Provisional Patent Application No. 63 / 448,655, filed February 27, 2023. Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted electronically in .xml format and is incorporated herein by reference in its entirety. The .xml copy, created on January 31, 2023, is referred to as Sequence Listing.xml and is 180,965 bytes in size. Field

[0003] This application relates to recombinant protein production in highly productive cells using serum-free media cultured for large-scale production, with improved systems and methods for measuring and maintaining cell culture conditions to harvest and purify proteins with higher yields, reduced heterogeneity, and improved quality. [Background technology]

[0004] Manufacturing processes for therapeutic protein products must be optimized according to factors including, but not limited to, protein production conditions, protein structural and functional properties, and the desired final drug product. Even when using the same therapeutic protein for the same final drug product, changes in protein production conditions may be necessary to optimize product titer, batch size, yield, or quality. Increasing and optimizing titer, batch size, and yield, in turn, requires optimizing harvest and purification processes to process increased titers and loads while maintaining acceptable quality attributes.

[0005] To support the increasing demand for recombinantly produced protein drug products, cell manufacturing processes have been developed that can produce higher titers and improved quality, including reduced heterogeneity and fewer sequence variants, while improving overall yield. Thus, there is a need for improved cell culture media that meet these demands and specialized cellular conditions.

[0006] Furthermore, measuring and maintaining optimized cell culture conditions is very important, which 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 affect the equipment used to measure cell culture conditions, including its accuracy and maintenance requirements. Because the biopharmaceutical industry is highly regulated, measurement irregularities may require investigation, potentially requiring additional raw materials and adding delays to 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 reduced maintenance requirements.

[0007] After production, isolation of highly concentrated proteins presents additional challenges, including process volume limitations, filtration capacity, chromatographic column loading capacity, and potentially high drug substance viscosity, each of which can affect outcomes such as material usage, time consumption, product quality, batch purity, and batch yield. Therefore, to address these challenges, novel approaches to harvesting, purifying, and formulating high-titer therapeutic proteins at each unit operation of an exemplary harvest and purification process have also been developed.

[0008] Improved methods and systems have been developed to manufacture, harvest, and purify higher titer antibody drug products to accommodate the processing of higher titer drug products. For example, increasing the titer of drug product and drug load can exceed the capacity of a particular chromatographic purification process and can also alter viscosity, which can negatively affect the transfer of the drug product along different chromatographic steps. Therefore, process improvements are needed that address not only individual purification steps, but the entire manufacturing process. Summary of the Invention

[0009] The present disclosure provides, in part, improved processes for large-scale production of anti-IL4Rα antibodies in highly productive cells. Improved cell culture media and processes for maintaining optimal cell culture conditions have been developed to increase titer and improve quality. Improved processes have also been developed to harvest and purify increased protein throughput while increasing overall yield, reducing manufacturing and operational complexity, and improving the overall quality of the drug substance and formulated drug product.

[0010] 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 complementarity determining region (HCDR) sequences comprising SEQ ID NOs: 3, 4, and 5, and three light chain complementarity determining region (LCDR) sequences comprising SEQ ID NOs: 6, 7, and 8.

[0011] 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 embodiments, the protein of interest is a protein having an Fc domain. Thus, in some embodiments, the protein of interest is an antibody, e.g., a human antibody, a humanized antibody, a chimeric antibody, an antibody fragment, e.g., a Fab or F(ab')2, an scFv molecule, etc.

[0012] In some embodiments, the manufacturing process includes culturing cells capable of expressing an anti-IL-4Rα antibody or a receptor antagonist as described above in a cell culture medium comprising a chemically defined base medium, such as a custom-formulated or commercially available base medium. In another embodiment, the complete medium is serum-free (a "serum-free" medium) and / or hydrolysate-free. In another embodiment, the method includes adding one or more point-of-use additives to the cell culture medium, as described below. In some embodiments, the point-of-use additives described below may also be included in the cell culture medium from the beginning.

[0013] Polyamine-supplemented media ("PS media") have been found to increase cell viability and density, decrease cell doubling time, and enable high-titer protein production by cells grown in such supplemented media. PS media, with or without serum and / or supplemented hydrolysates, have been found to provide restored cell viability and density, cell doubling time, and high-titer protein production.

[0014] In one embodiment, cells cultured in PS medium have an average doubling time of 30 hours or less. In one aspect, the cell doubling time is 24 hours or less. Similarly, the inclusion of polyamines in serum-free medium allows cultured cells to reach a higher viable cell density (VCD) than in the absence of polyamines.

[0015] In one embodiment, the medium comprises one or more polyamines selected from the group consisting of ornithine, putrescine, spermine, spermidine or combinations thereof in a serum-free and / or hydrolysate-free cell culture medium.

[0016] In one embodiment, the PS medium is serum-free and contains ornithine at a concentration of 30 μM to 900 μM. Ornithine may be present in the medium at a concentration (expressed in micromoles per liter) of 0, 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. Ornithine may be present at between about 0.09 mM and about 0.9 mM. In some embodiments, the PS medium contains ≦7.5 g / L hydrolysate. In some embodiments, the PS medium does not contain any hydrolysate.

[0017] In one embodiment, the PS medium is serum-free and contains putrescine at a concentration of 30 μM to 900 μM. In one aspect, the putrescine is 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, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 500, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 600, The putrescine may be present in the medium at a concentration (expressed in micromoles per liter) of 5, 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. Putrescine may be present at about 0.20 mM to about 0.714 mM. In one embodiment, the PS medium contains 57 mg / L ± 8.55 mg / L putrescine·2HCl in addition to ≥ 15 mg / L ± 2.25 mg / L ornithine·HCl. In some embodiments, the medium contains ≤ 7.5 g / L hydrolysate. In some embodiments, the PS medium does not contain any hydrolysate.

[0018] In one embodiment, the PS medium is serum-free and contains spermine at a concentration of 10 μM to 900 μM. In one aspect, spermine is present in an amount 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, 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). In some embodiments, the PS medium contains ≦7.5 g / L hydrolysate. In some embodiments, the PS medium does not contain any hydrolysate.

[0019] In one embodiment, the PS medium is serum-free and contains spermidine at a concentration of 10 μM to 900 μM. In one aspect, spermidine is present in an amount 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, 300, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 400, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 500, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 600, 61 The medium is present in a concentration (expressed in micromoles per liter) of 0, 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. In one embodiment, the PS medium contains ≥15 mg / L ±2.25 mg / L spermidine·HCl plus 57 mg / L ±8.55 mg / L spermine·4HCl. In some embodiments, the medium contains ≤7.5 g / L hydrolysate. In some embodiments, the medium contains ≤16 g / L hydrolysate. In some embodiments, the PS medium does not contain any hydrolysate.

[0020] In one embodiment, the medium comprises a mixture of amino acids (with the notable exception of glutamine, which can also be added back into the medium as a point-of-use additive) selected from the group 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.

[0021] In some embodiments, the medium comprises at least 40±6 mM or at least 70±10.5 mM of the mixture of amino acids or amino acid salts. In one embodiment, the medium comprises at least 40 mM of the mixture of amino acids. In this or another embodiment, the medium comprises at least 70 mM of the mixture of amino acids. In some embodiments, the total process, including the base medium and the feed, comprises a total of at least 115 mM of the mixture of amino acids or amino acid salts.

[0022] In some embodiments, the medium comprises one or more fatty acids. In one aspect, the medium comprises a mixture of fatty acids (or fatty acid derivatives) and alpha-tocopherol. In one aspect, the fatty acid or fatty acid derivative is selected from the group consisting of linoleic acid, linolenic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid, and may also include thioctic acid derived from fatty acids and combinations thereof.

[0023] In some embodiments, the 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.

[0024] In some embodiments, the cell culture medium comprises one or more salts. In some embodiments, the salts are selected from the group consisting of divalent cations, such as calcium, magnesium, and combinations thereof. In one embodiment, the medium comprises calcium chloride, magnesium sulfate, and combinations thereof. In another embodiment, the salts may also include phosphate salts.

[0025] In some embodiments, the cell culture medium comprises any one or more of NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4EDTA, Na citrate, and combinations thereof. In one aspect, the method employs adding one or more point-of-use chemicals selected from the group consisting of NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4EDTA, Na citrate, and combinations thereof to the cell culture medium.

[0026] In some embodiments, the point-of-use additives include taurine, phosphate, poloxamer 188, and combinations thereof. In some embodiments, the point-of-use additives may be included in the medium from the beginning.

[0027] Inclusion of taurine in cell culture media has been found to increase cell-specific productivity and allow for a reduction in ammonia by-production by these cells. In one embodiment, the cell culture medium is serum-free and contains 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.

[0028] In some embodiments, the present disclosure provides a method for producing an anti-IL-4Rα antibody or antigen-binding fragment thereof by using the following steps: (1) introducing into cells a nucleic acid sequence encoding a protein of interest; (2) selecting cells that harbor the nucleic acid sequence; (3) culturing the selected cells in an embodiment of a serum-free cell culture medium described in the present disclosure; and (4) expressing the protein of interest in the cells, wherein the protein of interest is secreted into the medium. The biotherapeutic protein may be a blocking antibody or receptor antagonist (e.g., blocking IL-4Rα and IL-13) and / or an antigen-binding protein and may include an Fc domain. In some embodiments, the antibody is a human, humanized, chimeric, or non-human monoclonal antibody or antibody fragment.

[0029] In some embodiments, the cell or cells are mammalian cells, avian cells, insect cells, yeast cells, or bacterial cells. In one embodiment, the cells are mammalian cells useful for producing recombinant proteins, such as CHO cells or derivatives thereof, or CHO-K1 cells. In some embodiments, the cells express a protein of interest, such as a biotherapeutic protein. In some embodiments, the cells used for protein production are mammalian cells capable of producing biotherapeutic agents, such as CHO, HEK293, and BHK cells, or any derivatives thereof. In one embodiment, the cells are CHO cells, such as CHO-K1 cells.

[0030] 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; and (4) comprises at least about 40 mM or at least a mixture of amino acids comprising 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) containing about 70 mM tocopherol and a fatty acid mixture; (6) containing a mixture of nucleosides including at least one of the following: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; (7) containing at least one salt of calcium, magnesium, and phosphate; and (8) containing at least one of the following point-of-use additives: NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4EDTA, Nacitrate, and combinations thereof.

[0031] In some embodiments, the cell culture medium (1) is serum-free; (2) comprises one or more polyamines, including at least ornithine or spermine; (3) comprises at least about 40 mM or at least about 70 mM of a mixture of amino acids comprising 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; and (4) (5) a mixture of nucleosides comprising at least one of the following: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; (6) a salt comprising at least one of the following: calcium, magnesium, phosphate, and combinations thereof; and (7) at least one of the following point-of-use additives: NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4EDTA, Nacitrate, and combinations thereof.

[0032] In some embodiments, the cell culture medium (1) is serum-free; (2) comprises ≦7.5 g / L of hydrolysate; (3) comprises 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM ornithine; (4) optionally comprises 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) comprises alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, (5) comprising at least about 40 mM or at least about 70 mM of a mixture of amino acids comprising at least one of isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof; (6) comprising tocopherol and optionally a mixture of fatty acids; (7) comprising a mixture of nucleosides comprising adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; and (8) comprising salts of calcium, magnesium, phosphate, and combinations thereof.

[0033] In some embodiments, the cell culture medium (1) is hydrolysate-free; (2) contains ornithine at a concentration of at least 90 μM ± 14 μM; and (3) optionally contains putrescine, such as at least 150 μM ± 14 μM. In other embodiments, other polyamines, such as spermine, spermidine, etc., are contemplated within the scope of the present invention.

[0034] In some embodiments, the present disclosure provides a method for the preparation of a soluble fiber comprising: (1) any of 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM ornithine; (2) optionally, further, any of 0.20±0.03 mM, 0.35±0.06, or 0.714±0.11 mM putrescine; (3) about 40 mM or at least about 70 mM of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, thiamin ... The present invention provides a method for culturing cells in a serum-free medium containing: (4) an amino acid mixture containing onine, tryptophan, tyrosine, and valine; (5) a tocopherol and fatty acid mixture; (6) a nucleoside mixture containing adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine; and (7) calcium, magnesium, and a phosphate salt, wherein the cells cultured by this method have an average doubling time of 24 hours or less, or one-third or less than the doubling time of cells cultured using a similar cell culture medium containing less than 0.09±0.015 mM polyamines. In another embodiment, the cell culture is capable of achieving a viable cell density at least three times greater than that of 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 medium comprises ≦7.5 g / L hydrolysate; in another embodiment, the medium is hydrolysate-free.

[0035] In some embodiments, the protein of interest is prepared by (1) introducing a nucleic acid sequence encoding the protein of interest, e.g., an antibody or other antigen-binding protein, into CHO cells; (2) selecting cells harboring the nucleic acid sequence; and (3) adding at least one of: (a) 0.09±0.014, 0.3±0.05, 0.6±0.09, or 0.9±0.14 mM ornithine; (b) optionally, at least 0.20±0.03, 0.35±0.06, or 0.714±0.11 mM putrescine; (c) at least 40 mM or at least 70 mM of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, rhodium ... The protein of interest is produced by culturing selected cells in a serum-free cell culture medium containing: (a) an amino acid mixture including one or more of stidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (b) tocopherol and, optionally, a fatty acid mixture; (c) a nucleoside mixture including one or more of adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; and (d) salts including one or more of calcium, magnesium, phosphate, and combinations thereof; and (e) expressing the protein of interest in the CHO cells, wherein the protein of interest is secreted into the medium. In some embodiments, the serum-free cell culture medium may contain ≦7.5 g / L of hydrolysate, and in other embodiments, may contain no hydrolysate at all.

[0036] In some embodiments, the protein of interest is prepared by (1) introducing into CHO cells a nucleic acid sequence encoding the protein of interest, e.g., an antibody or other antigen-binding protein; (2) selecting cells harboring the nucleic acid sequence; and (3) selecting the cells harboring the nucleic acid sequence and (4) selecting the cells harboring the nucleic acid sequence and (5) selecting the cells harboring the nucleic acid sequence and (6) selecting the cells harboring the nucleic acid sequence and (7) selecting the cells harboring the nucleic acid sequence and (8) selecting the cells harboring the nucleic acid sequence and (9) selecting the cells harboring the nucleic acid sequence and (10) selecting the cells harboring the nucleic acid sequence and (11) selecting the cells harboring the nucleic acid sequence and (12) selecting the cells harboring the nucleic acid sequence and (13) selecting the cells harboring the nucleic acid sequence and (14) selecting the cells harboring the nucleic acid sequence and (15) selecting the cells harboring the nucleic acid sequence and (16) selecting the cells harboring the nucleic acid sequence and (17) selecting the cells harboring the nucleic acid sequence and (18) selecting the cells harboring the nucleic acid sequence and (19) selecting the cells harboring the nucleic acid sequence and (20) selecting the cells harboring the nucleic acid sequence and (21) selecting the cells harboring the nucleic acid sequence and (22) selecting the cells harboring the nucleic acid sequence and (23) selecting the cells harboring the nucleic acid sequence and (24) selecting the cells harboring the nucleic acid sequence and (25) selecting the cells harboring the nucleic acid sequence and (26) selecting the cells harboring the nucleic acid sequence and (27) selecting the cells harboring the nucleic acid sequence and (28) selecting the cells harboring the nucleic acid sequence and (29) selecting the cells harboring the nucleic acid sequence and (30) selecting the cells harboring the nucleic acid sequence and (31) selecting the cells harboring the (d) a mixture of amino acids including one or more of: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof; (d) a tocopherol and fatty acid mixture; (e) a mixture of nucleosides including one or more of: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; and (f) salts including one or more of calcium, magnesium, and phosphate; and (3) expressing the protein of interest in the CHO cells, wherein the protein of interest is secreted into the medium. In some embodiments, the serum-free cell culture medium may include ≦7.5 g / L of hydrolysate, or in other embodiments, no hydrolysate at all.

[0037] The present disclosure provides, in part, an improved seed train process for increasing titer. In one embodiment, the initial viable cell density (VCD) at each step (N-5 to N-1) is 1.7x10 5 ~2.3x10 5 Compared to standard initial VCDs in the range of cells / mL, approximately 3.5x10 5 ~Approx. 5.43x10 5cells / mL. In some embodiments, the initial VCD (N-5 to N-1) is about 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, or 3.0x greater than the alternative initial VCD in a standard seed train. In one embodiment, the optimized seed train resulted in 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 embodiment, there is no substantial difference in peak lactate observed in the final production vessel compared to a standard seed train.

[0038] The present disclosure provides, in part, improved vessels 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.

[0039] The present disclosure provides, in part, improved methods and systems for aeration, agitation, and dissolved gas measurement. More specifically, the present disclosure provides improved bioreactors with optimized agitation rates and working volumes to provide the necessary agitation while minimizing shear stress, as well as providing optimized dissolved gas concentrations at various intervals.

[0040] In certain exemplary embodiments, starting volumes, air sparging, and agitation set points in vessels and bioreactors containing various sensors along the seed train during the growth and production phases are optimized to improve process robustness through minimizing shear stress on the cell culture and optimizing viable cell density (VCD), dissolved oxygen, carbon dioxide, and pH at various time points.

[0041] In some embodiments, the aeration rate may be used to alter the pCO2 level within the cell culture to modulate the acidic and basic charge variants for cultured cells expressing dupilumab.

[0042] The present disclosure also provides improved methods and systems for measuring dissolved gases. Electrochemical and optical probes may also be used to measure other dissolved gases, such as carbon dioxide. Electrochemical probes have a long history of exhibiting a wider linear range and faster response time compared to optical probes or sensors, and therefore have been more commonly used in vessels and bioreactors than optical probes. However, electrochemical dissolved oxygen probes require significant maintenance costs due to frequent replacement of membranes and electrolyte solutions, as well as longer polarization times. Furthermore, electrochemical probes are known to provide noisy readings due to bubble interference; evidence of this occurrence in large-scale bioreactors is provided in the following examples. Such noise can cause excursions outside the normal operating range, which must be investigated and documented through biopharmaceutical companies' quality compliance systems due to the high level of regulation within the industry. Such short excursions, typically on the high side due to bubble interference, are investigated and result in a cost to the manufacturing raw materials.

[0043] Optical probes are less affected by bubble interference from stirring, agitation, and aeration, and do not have the same electrolyte exchange and polarization requirements; however, optical probes are less accurate and less reactive than electrochemical probes. Furthermore, previous studies evaluating the use of optical probes have been limited to water monitoring and small-scale cell culture applications. The largest vessel used in the reviewed literature during optical probe testing was a 50 L single-use 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 (>1,000 L) that does not address performance issues with optical probes.

[0044] Thus, the present disclosure provides improved systems and methods for reducing signal noise in electrochemical probes, reducing or eliminating false positives that can result from bubble interference. Furthermore, the present disclosure provides systems and methods for improving the accuracy of optical probes used to measure dissolved gases in large-scale vessels and bioreactors, which require lower maintenance and are less susceptible to bubble interference than conventional electrochemical probes. As discussed in more detail below, vessels and bioreactors containing electrochemical and optical probes have been developed with improved data processing methods to optimize measurement accuracy while minimizing maintenance by reducing or eliminating undesirable excursion events outside of normal operating ranges that require lengthy quality inspections in the highly regulated biopharmaceutical industry.

[0045] In one embodiment, the present disclosure provides a method for measuring the VCD of cells cultured in a bioreactor by applying an electric field to the cells cultured in the bioreactor, measuring the capacitance, and correlating the measured capacitance to viable cell density.

[0046] In one embodiment, the protein of interest can be produced at an average 7-day titer that is at least 7% greater, at least 14% greater, at least 80% greater, at least two-fold greater, or at least three-fold greater than the average 7-day titer produced by similar cells in serum-free 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).

[0047] New and improved methods have been developed for harvesting and purifying increased protein production that not only provide increased protein production but also improve overall yield, including, for example, pre-harvest optimization, Protein A chromatography, viral inactivation, anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, viral retentive filtration, concentration and diafiltration, and drug substance modification, which are further described below.

[0048] The present disclosure provides methods for purifying anti-IL4Rα antibodies. In some exemplary embodiments, the methods include (a) subjecting the antibodies to pretreatment, (b) harvesting the antibodies, (c) subjecting the antibodies to affinity chromatography, (d) subjecting the pooled antibodies 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 pooled antibodies from step (d) to anion exchange chromatography in flow-through mode, (f) subjecting the pooled antibodies from the flow-through fraction of step (e) to cation exchange chromatography in bind-and-elute mode, (g) subjecting the pooled antibodies from the eluate of step (f) to hydrophobic interaction chromatography in flow-through mode, and (h) subjecting the pooled antibodies from the flow-through fraction of step (g) to virus-retentive filtration.

[0049] In one embodiment, the antibody of step (h) is further subjected to concentration and diafiltration using a diafiltration buffer having a pH of 4.0 to 4.5.

[0050] In one embodiment, the diafiltration buffer comprises about 4 mM acetic acid to about 6 mM acetic acid.

[0051] In one embodiment, the pre-harvest treatment includes subjecting the antibody to a transient 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.

[0052] In one embodiment, the affinity chromatography is Protein A chromatography.

[0053] In one embodiment, the harvesting in step (b) includes centrifugation and depth filtration.

[0054] In one embodiment, the antibody is harvested in a 3 kL to 25 kL bioreactor.

[0055] In one embodiment, the Protein A column loading pH is 7 to 8. In one embodiment, the Protein A column loading pH is 6 to 8. In one embodiment, the Protein A column loading pH is about 6.

[0056] In another exemplary embodiment, the method includes the steps of: (a) culturing cells expressing an anti-IL-4Rα antibody or antigen-binding fragment thereof; (b) subjecting the cells to a transient pH level of about 4.5 to 5.0, or about 4.0 to about 5.5, and then adjusting the pH to about 5.5 to 6.5; (c) harvesting the cells by centrifugation and separating cellular debris from the clarified medium containing the anti-IL-4Rα antibody or antigen-binding fragment thereof; (d) subjecting the clarified medium to affinity chromatography; (e) subjecting the anti-IL-4Rα antibody or antigen-binding fragment thereof pooled from the eluate of step (d) to viral inactivation at a pH of about 3 to about 4.5, and then adjusting the pH to about 5 to about 8; and (f) (g) subjecting the pooled anti-IL-4Rα antibodies or antigen-binding fragments thereof from step (e) to anion exchange chromatography in flow-through mode; (g) subjecting the pooled anti-IL-4Rα antibodies or antigen-binding fragments thereof from the flow-through fraction of step (f) to cation exchange chromatography in bind and elute mode; (h) subjecting the pooled anti-IL-4Rα antibodies or antigen-binding fragments thereof from the eluate of step (g) to hydrophobic interaction chromatography in flow-through mode; and (i) subjecting the pooled anti-IL-4Rα antibodies or antigen-binding fragments thereof from the flow-through fraction of step (h) to virus-retentive filtration, thereby producing the anti-IL-4Rα antibodies or antigen-binding fragments thereof.

[0057] In one embodiment, the method further comprises, after step (i), subjecting the anti-IL-4Rα antibody or antigen-binding fragment thereof to ultrafiltration and diafiltration (UF / DF).

[0058] In one embodiment, the affinity chromatography is Protein A chromatography.

[0059] In one embodiment, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining region (HCDR) sequences comprising SEQ ID NOs: 3, 4, and 5, and three light chain complementarity determining region (LCDR) sequences comprising SEQ ID NOs: 6, 7, and 8.

[0060] In one embodiment, 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.

[0061] In one embodiment, the anti-IL-4Rα antibody is dupilumab.

[0062] These and other aspects of the present disclosure will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments and many specific details thereof, is offered by way of illustration and not limitation. Various substitutions, modifications, additions, or rearrangements may be made within the scope of the present invention, and further numbered examples are provided below that describe further aspects of the present disclosure. [Brief explanation of the drawings]

[0063] [Figure 1] FIG. 1 illustrates the structure of the antibody product, dupilumab, according to an exemplary embodiment.

[0064] [Figure 2] FIG. 2 shows the effect of insulin replacement on the potency profile of dupilumab, according to an exemplary embodiment.

[0065] [Figure 3] FIG. 3 shows the effect of insulin supplementation on the viability of cells expressing dupilumab, according to an exemplary embodiment.

[0066] [Figure 4]FIG. 4 shows the effect of insulin supplementation on reducing ammonia levels in cultures containing cells expressing dupilumab, according to an exemplary embodiment.

[0067] [Figure 5] FIG. 5 shows the effect of 15 mg / L insulin concentration and feeding schedule on growth rate, upper asymptote (highest achievable titer), inflection point, and titer of the titer curve, according to an exemplary embodiment.

[0068] [Figure 6] FIG. 6 shows the effect of insulin replacement using a profiler on day 13 on high molecular weight species, according to an exemplary embodiment.

[0069] [Figure 7] FIG. 7 shows the effect of insulin supplementation using the profiler on day 13 on non-reduced purity levels, according to an exemplary embodiment.

[0070] [Figure 8] FIG. 8 shows the effect of insulin replacement schedules using the profiler on day 13 on non-glycosylated heavy chain (NGHC) levels, according to an exemplary embodiment.

[0071] [Figure 9] FIG. 9 shows the effect of tyrosine supplementation on titer and ammonia levels in cell cultures expressing dupilumab, according to an exemplary embodiment.

[0072] [Figure 10] FIG. 10 shows the effect of phosphate redistribution strategies 6 and 8 on dupilumab potency (g / L), according to an exemplary embodiment.

[0073] [Figure 11] FIG. 11 shows phosphate redistribution profiles on days 0, 2, 4, 6 and 8 according to an exemplary embodiment.

[0074] [Figure 12] FIG. 12A shows a contour plot of a central composite design (CCD) model predicting host cell protein clearance coefficient (CF), according to an exemplary embodiment.

[0075] FIG. 12B shows a contour plot of a CCD model predicting normalized supernatant turbidity (NTU), according to an exemplary embodiment.

[0076] [Figure 13] FIG. 13A shows a contour plot of a CCD model predicting host cell protein clearance coefficient, according to an illustrative embodiment.

[0077] FIG. 13B shows a contour plot of a CCD model predicting normalized supernatant turbidity (NTU), according to an exemplary embodiment.

[0078] [Figure 14] FIG. 14 illustrates the initial and final viable cell density (VCD) for the average standard and optimized seed trains, according to an exemplary embodiment, with the optimized seed train having an increased initial VCD compared to the standard seed train.

[0079] [Figure 15] FIG. 15 shows biomass reduction in a 10 kL production bioreactor for cells cultured using an optimized seed train with increased initial VCD compared to a standard seed train, according to an exemplary embodiment.

[0080] [Figure 16] 16A and 16B show the final titer (g / L) of an optimized seed train (Sample 1) with increased initial VCD compared to a standard seed train (Samples 2-9) according to an exemplary embodiment.

[0081] [Figure 17]FIG. 17 shows a bivariate linear regression of online capacitance and offline VCD measurements of Cell Line A (CLA) in a seed train, according to an exemplary embodiment of the present invention.

[0082] [Figure 18] FIG. 18 shows a bivariate linear regression of online capacitance and offline VCD measurements of CLA during seed train phases N-3, N-2, and N-1, according to an exemplary embodiment of the present invention.

[0083] [Figure 19] Figure 19 shows predictions of online VCD measurements of CLA during seed train N-3, N-2, and N-1 phases, according to an exemplary embodiment of the invention, as determined using online capacitance measurements and the correlation equation presented in Figure 21. The dots (circles) indicate VCD values ​​determined offline using a bioanalyzer.

[0084] [Figure 20] FIG. 20 shows a bivariate linear regression of online capacitance and offline VCD measurements of Cell Line B (CLB) in a seed train, according to an exemplary embodiment of the present invention.

[0085] [Figure 21] FIG. 21 shows a bivariate linear regression of online capacitance and offline VCD measurements of CLB during seed train phases N-3, N-2, and N-1, according to an exemplary embodiment of the present invention.

[0086] [Figure 22] Figure 22 shows predictions of online VCD measurements of CLB during seed train N-3, N-2, and N-1 phases, according to an exemplary embodiment of the present invention, as determined using online capacitance measurements and the correlation equation presented in Figure 24. The dots (circles) indicate VCD values ​​determined offline using a bioanalyzer.

[0087] [Figure 23] FIG. 23 shows a bivariate linear regression of online capacitance and offline VCD measurements of CLA and CLB in a seed train according to an exemplary embodiment of the present invention.

[0088] [Figure 24] 24A, 24B, and 24C show predictions of online VCD measurements of CLA during seed train N-3, N-2, and N-1 phases, according to an exemplary embodiment of the invention, as determined using the online capacitance measurements presented in Figure 17 and the correlation equations presented in Figures 17 (Figure 24A), 20 (Figure 24B), and 23 (Figure 24C). The dots (circles) indicate VCD values ​​determined offline using a bioanalyzer.

[0089] [Figure 25] 25A, 25B, and 25C show predictions of online VCD measurements of CLB during seed train N-3, N-2, and N-1 stages (black lines) as determined using the online capacitance measurements presented in FIG. 21 and the correlation equations presented in FIG. 20 (FIG. 25A), FIG. 17 (FIG. 25B), and FIG. 23 (FIG. 25C), according to an exemplary embodiment of the invention. The dots (circles) indicate VCD values ​​determined offline using a bioanalyzer.

[0090] [Figure 26] Figures 26A and 26B show predictions of online VCD measurements of CLBs during the seed train and production phases, determined using the online capacitance measurements and correlation equation presented in Figure 20, according to an exemplary embodiment of the invention. Panels A and B each show data for two separate cultures using a single probe to perform the online capacitance measurements. In Figure 26A, the line shows the prediction of the online VCD measurements, and the dots (circles) show the VCD values ​​determined offline using a bioanalyzer. In Figure 26B, the line shows the prediction of the online VCD measurements, and the dots (circles) show the VCD values ​​determined offline using a bioanalyzer.

[0091] [Figure 27] FIG. 27A shows host cell protein values ​​and dupilumab yield after acid precipitation of dupilumab cell culture using 1 M phosphoric acid, according to an exemplary embodiment.

[0092] FIG. 27B shows host cell protein values ​​and dupilumab yield after acid precipitation of dupilumab cell culture medium using 1.75 M acetic acid, according to an exemplary embodiment.

[0093] [Figure 28] FIG. 28 shows an exemplary schematic diagram of the use of depth filters, polish filters and guard filters according to an exemplary embodiment.

[0094] [Figure 29] FIG. 29 illustrates a predictive profiler modeling factors and responses studied in a centrifugation characterization study, according to an exemplary embodiment.

[0095] [Figure 30] FIG. 30 shows a schematic diagram of an exemplary refinement step of the optimization process, according to an exemplary embodiment.

[0096] [Figure 31] FIG. 31 shows a schematic diagram of an exemplary refinement step of the optimization process, according to an exemplary embodiment.

[0097] [Figure 32] FIG. 32 shows a visualization of a model generated for a multivariate study of affinity capture and viral inactivation risk factors and responses, according to an exemplary embodiment.

[0098] [Figure 33] FIG. 33 shows HCP levels in the affinity capture pool using various affinity wash buffers, according to an exemplary embodiment.

[0099] [Figure 34] FIG. 34 shows HCP levels after AEX or HIC processing downstream of affinity capture chromatography using various affinity wash buffers, according to an exemplary embodiment.

[0100] [Figure 35] FIG. 35 shows protein yield and HMW species in the affinity capture pool using various affinity wash buffers, according to an exemplary embodiment.

[0101] [Figure 36] FIG. 36 shows a visualization of a model generated for a multivariate study of AEX risk factors and response, according to an exemplary embodiment.

[0102] [Figure 37] FIG. 37 shows a visualization of a model generated for a multivariate study of CEX risk factors and response, according to an exemplary embodiment.

[0103] [Figure 38] FIG. 38 shows a visualization of a model generated for a multivariate study of HIC risk factors and response, according to an exemplary embodiment.

[0104] [Figure 39] FIG. 39 shows a visualization of a model generated for a multivariate study of VRF risk factors and response, according to an exemplary embodiment.

[0105] [Figure 40] FIG. 40 shows a visualization of a model generated for a multivariate study of UF / DF risk factors and response, according to an exemplary embodiment.

[0106] [Figure 41]FIG. 41 shows bioreactor titers for a dupilumab 500 L validation batch compared to a 10,000 L process performance qualification batch and a 2 L mirror production, according to an exemplary embodiment.

[0107] [Figure 42] FIG. 42 shows step yields by unit operation for a 500 L validation batch compared to a 10,000 L process performance qualification batch, according to an exemplary embodiment.

[0108] [Figure 43] FIG. 43 shows HMW content by unit operation for a 500 L validation batch compared to a 10,000 L process performance qualification batch, according to an exemplary embodiment.

[0109] [Figure 44] Figure 44 shows HCP content by unit operation for a 500 L validation batch compared to a 10,000 L process performance qualification batch, according to an exemplary embodiment.

[0110] [Figure 45] FIG. 45 illustrates an example of stepwise agitation and aeration rates over time according to an exemplary embodiment.

[0111] [Figure 46] 46 illustrates an example of signal filtering for a cell culture with a 59 second sampling rate and approximately 10 minute sample window with an agile filter applied, according to an exemplary embodiment. Label "A" illustrates that low-side excursions remain in the smoothed signal. Label "B" illustrates excellent agreement between the smoothed and unfiltered signals for expected process disturbances.

[0112] [Figure 47]47 illustrates an example of signal filtering for a cell culture with a 59 second sampling rate and approximately 33 minute sample window with an agile filter applied, according to an exemplary embodiment. Label "A" illustrates that low side excursions are removed in the smoothed signal. Label "B" illustrates excellent agreement between the smoothed and unfiltered signals for expected process disturbances.

[0113] [Figure 48] 48 illustrates an example of signal filtering for a cell culture at a 59 second sampling rate and approximately 33 minute sample window with a Savitsky Golay filter applied, according to an exemplary embodiment. Label "A" illustrates the remaining noise for the filtered signal. Label "B" illustrates the excellent agreement between the smoothed and unfiltered signals for expected process disturbances.

[0114] [Figure 49] FIG. 49 illustrates a stack comparison of the filtered signals of FIGS. 46-48, according to an exemplary embodiment.

[0115] [Figure 50] FIG. 50 illustrates the dissolved oxygen trend for Run 1 (lightly shaded line is electrochemistry, medium shaded line is optical probe B, darkest shaded line is optical probe A, dotted lines: setpoint and upper and lower normal operating range limits) according to an exemplary embodiment.

[0116] [Figure 51] FIG. 51 illustrates the dissolved oxygen trend for Run 2 (lightly shaded line is electrochemistry, medium shaded line is optical probe B, darkest shaded line is optical probe A, dotted lines: setpoint and upper and lower normal operating range limits), according to an exemplary embodiment.

[0117] [Figure 52]FIG. 52 illustrates the dissolved oxygen trend for Run 3 (lightly shaded line is electrochemical, medium shaded line is optical probe B, darkest shaded line is optical probe A, dotted lines: setpoint and upper and lower normal operating range limits), according to an exemplary embodiment.

[0118] [Figure 53] FIG. 53 illustrates the dissolved oxygen trend for Run 4 (lightly shaded line is electrochemistry, medium shaded line is optical probe B, darkest shaded line is optical probe A, dotted lines: setpoint and upper and lower normal operating range limits), according to an exemplary embodiment.

[0119] [Figure 54] FIG. 54 illustrates dissolved oxygen data generated using Optical Probe B with an anti-bubble cap for Runs 1-3 and a standard cap for Run 4, in accordance with an exemplary embodiment.

[0120] [Figure 55] FIG. 55 illustrates dissolved oxygen data (lightly shaded line is electrochemistry, medium shaded line is optical probe B, darkest shaded line is optical probe A, bottom line is electrochemistry with agile filter (59 second sampling rate and default sample window)) as determined at time segments at the "beginning," "middle," and "end" of Run 2 examined as part of offset evaluation, according to an exemplary embodiment.

[0121] [Figure 56] FIG. 56 illustrates Run 1 Probe B offset correction (medium shaded line is Optical Probe B, darkest shaded line is Optical Probe B adjusted upward by 3.0%, lightly shaded line is Electrochemistry with Agile Filter (59 sec sampling rate and default sample window), dotted lines: setpoint and upper and lower normal operating range limits) according to an exemplary embodiment.

[0122] [Figure 57]FIG. 57 illustrates Run 3 Probe B offset correction (medium shaded line is Optical Probe B, darkest shaded line is Optical Probe B adjusted upward by 3.0%, lightly shaded line is Electrochemistry with Agile Filter (59 sec sampling rate and default sample window), dotted lines: set point and upper and lower normal operating range limits) according to an exemplary embodiment.

[0123] [Figure 58] Figures 58A and 58B illustrate examples of various dextrose concentrations at various time points.

[0124] [Figure 59] 59 illustrates the pCO2 profiles of culture media at various aeration conditions in mmHg (y-axis) over process time (days), according to an exemplary embodiment. A moderate pCO2 was selected as the midpoint control.

[0125] [Figure 60] FIG. 60 shows a visualization of a model generated for a multivariate study of mixed-mode chromatography (MMC) risk factors and responses using Capto Adhere resin, according to an exemplary embodiment.

[0126] [Figure 61] FIG. 61 shows a visualization of a model generated for a multivariate study of MMC risk factors and response using PPA HyperCel resin, according to an exemplary embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0127] The manufacturing process for therapeutic protein products, such as monoclonal antibody drugs, must be optimized according to factors including, but not limited to, recombinant host cell characteristics, protein production conditions, protein structural and functional properties, and the desired final drug product. To support the increasing demand for recombinantly produced protein drug products, cell lines and cell culture media have been developed that are capable of producing higher titers and larger batch sizes with improved quality and yield.

[0128] Even when using the same therapeutic protein for the same final drug product, it may be necessary to vary protein production conditions to optimize titer, batch size, yield, or product quality. Applicants have discovered that adding one or a combination of polyamines, e.g., ornithine, putrescine, spermine, and spermidine, to culture media ("PS medium") improves viable cell density, cell doubling time, and protein production of dupilumab by cells in cell culture compared to serum-free media containing very little or no polyamines, e.g., ornithine, putrescine, spermine, and spermidine ("non-PS medium").

[0129] Conventional methods for harvesting and purifying protein drug products rely on a series of filtration and chromatography steps, which can be limited in their ability to process increased titers and corresponding batch sizes, protein concentrations, and drug product viscosities using similarly sized equipment while maintaining purity and quality standards. For example, pumps previously used to move drug products may fail to move drug products at higher protein concentrations due to increased viscosity levels. Techniques have been developed to reduce sample viscosity, using the addition of hydrophobic salts, such as arginine. However, adding arginine during harvesting and purification, especially during concentration and diafiltration, can result in the loss of unknown amounts of arginine in later processing steps, which must then be measured and compensated for further downstream, potentially increasing the variability of the final drug product. Therefore, there is a need for systems and methods for harvesting high-titer antibody drug products while maintaining product consistency, high yield, and quality.

[0130] The disclosure herein provides improvements to manufacturing, harvesting and purification processes to improve the production of high-titer antibody products. In some exemplary embodiments, the high-titer antibody product of interest is dupilumab.

[0131] Dupilumab is a human monoclonal immunoglobulin G4 (IgG4) antibody expressed and prepared from Chinese hamster ovary cells (CHO-K1) that targets the interleukin-4 receptor subunit alpha (IL-4Rα). After cell culture, harvesting, isolation, and purification, dupilumab is formulated for administration with, for example, histidine, histidine hydrochloride, arginine hydrochloride, polysorbate 80, sodium acetate, glacial acetic acid, sucrose, and water.

[0132] Dupilumab inhibits interleukin-4 (IL-4) and interleukin-13 (IL-13) signaling by specifically binding to IL-4 receptor alpha (IL-4Rα), which is shared by the interleukin-4 (IL-4) and interleukin-13 (IL-13) receptor complexes. Dupilumab inhibits IL-4 signaling through its type I receptor and both IL-4 and IL-13 signaling through its type II receptor. Blocking IL-4Rα with dupilumab inhibits IL-4- and IL-13-induced inflammatory responses, including the release of proinflammatory cytokines, chemokines, nitric oxide, and IgE. By blocking an essential shared component of the IL-4 / IL-13 receptor complex, dupilumab inhibits IL-4 and IL-13 signaling, a key disease driver of type II inflammation, providing clinical benefit and long-term disease control without the side effects commonly observed with current nonselective systemic immunosuppressants.

[0133] Dupilumab received its first global market authorization for the treatment of moderate to severe atopic dermatitis (AD) in adults in the United States on March 28, 2017, followed by approval in the European Union on September 28, 2017, in Japan on January 19, 2018, and subsequently in numerous 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 polyposis (CRSwNP), eosinophilic esophagitis (EoE), chronic spontaneous urticaria (CSU), and prurigo nodularis (PN). Additionally, subcutaneous administration of dupilumab is currently in development for the treatment of patients with type 2 inflammatory diseases, including AD (pediatric patients 6 months to <6 years of age), asthma (pediatric patients 6 months to <6 years of age), chronic obstructive pulmonary disease (COPD), allergic fungal rhinosinusitis (AFRS), chronic rhinosinusitis without nasal polyps (CRSsNP), allergies (e.g., grass allergy, peanut allergy, and dairy allergy), bullous pemphigoid, atopic dermatitis of the hands and feet, cold-induced urticaria, chronic urticaria, ulcerative colitis, chronic pruritus of unknown etiology, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, bronchiectasis, alopecia areata, and allergic rhinitis.

[0134] To meet the high demand for dupilumab, a novel manufacturing process was developed, as described further below, that allows for the production and purification of dupilumab 150 mg / mL and 175 mg / mL formulated drug substance (FDS) at, for example, 10,000 L and 25,000 L scales. Explanation of terms

[0135] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein that are known to those skilled in the art may be used in the practice of specific embodiments described herein. All publications mentioned are incorporated herein by reference in their entirety.

[0136] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard deviation 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 meant to be open-ended and are understood to mean "comprise," "comprises," and "comprising," respectively.

[0137] As used herein, the term "upstream processing technology" refers to activities involving protein production from cells in cell culture in the context of protein preparation. As used herein, the term "cell culture" refers to methods and techniques for generating and maintaining a host cell population capable of producing a recombinant protein of interest, as well as for optimizing the production and harvest of the protein of interest. For example, once an expression vector has been introduced into a suitable host cell, the host cell can be maintained under conditions suitable for expression of the appropriate nucleotide coding sequence, and the harvest and production of the desired recombinant protein.

[0138] The present disclosure provides serum-free media useful in culturing cells and producing biopharmaceutical drug substances. "Serum-free" refers to cell culture media that do not contain animal serum, e.g., fetal bovine serum. The serum-free media may contain ≦7.5 g / L of a hydrolysate, e.g., a soy hydrolysate. The present disclosure also provides chemically defined media (CDMs) that are not only serum-free but also hydrolysate-free.

[0139] "Hydrolysate-free" refers to a cell culture medium that does not contain any exogenous protein hydrolysates, e.g., animal or plant protein hydrolysates, including, for example, peptone, tryptone, and the like.

[0140] "Cell culture" or "culturing" refers to the growth and proliferation of cells outside a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See, for example, Animal Cell Culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid support. Fluidized-bed bioreactors, hollow-fiber bioreactors, roller bottles, flasks, or stirred-tank bioreactors, with or without microcarriers and operated in batch, fed-batch, continuous, semi-continuous, or perfusion modes, are available for mammalian cell culture. Cell culture medium or concentrated feed medium may be added to the culture continuously or at intervals during cultivation. For example, the culture may be fed once daily, every other day, every third day, or when the concentration of a particular monitored medium component falls outside the desired range.

[0141] As used herein, the terms "chemically defined medium" (both abbreviated "CDM") refer to synthetic growth media in which the identity and concentration of all components are defined. Chemically defined media do not contain bacteria, yeast, animal, or plant extracts, animal serum, or plasma, although individual plant- or animal-derived components (e.g., proteins, polypeptides, etc.) may be added. Chemically defined media may contain inorganic salts, such as phosphate, sulfate, etc., necessary to support growth. The carbon source is defined and is usually a sugar, such as glucose, lactose, galactose, etc., or other compounds, such as glycerol, lactate, acetate, etc. Certain chemically defined culture media also use phosphate as a buffer, while other buffers, such as sodium bicarbonate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), citrate, triethanolamine, etc., may also be used. 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, Inc.), various EX-CELLs media (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various IS CHO-CD media (FUJIFILM Irvine Scientific), combinations thereof, etc. Methods for preparing chemically defined culture media are known in the art and can be found, for example, in U.S. Patent 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 disclosures of which are incorporated herein by reference.

[0142] As used herein, the term "recombinant host cell" (or simply "host cell") includes a cell into which a recombinant expression vector encoding a protein of interest has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In one embodiment, host cells include prokaryotic and eukaryotic cells selected from any kingdom of life. In one aspect, eukaryotic cells include protist, fungal, plant, and animal cells. In a further aspect, host cells include eukaryotic cells, e.g., plant and / or animal cells. The cells can be mammalian, fish, insect, amphibian, or avian cells. In a particular 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 depositories, as well as commercial suppliers. Cells that may be used in the process 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 (ATCC CCL-61) (Chasin et al., 1986, Som. Cell Molec. Genet., 12:555-556; Kolkekar et al., 1997, Biochemistry, 36:10901-10909; and WO 01 / 92337 A2), 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); SV40-transformed monkey kidney CV1 cells (COS cells, COS-7, ATCC CRL-1651);HEK293 cells, Sp2 / 0 cells, 5L8 hybridoma cells, Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkat cells, THP-1 cells, Sp2 / 0 cells, primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells, and retinal epithelial cells) and established cell lines and their lineages (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 Sertoli 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, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-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, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH3 cells, L2 cells, LLC-RC 256 cells, MH1C1 cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof), fibroblasts derived 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 (arteries, veins, capillaries), lymphatic tissue (lymph glands, adenoids, tonsils, bone marrow, and blood), spleen), and fibroblast 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, MiCl1 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, C3H / IOTI / 2 cells, HSDM1C3 cells, KLN2 05 cells, McCoy cells, mouse L cells, 2071 strain (mouse L) cells, LM strain (mouse L) cells, L-MTK (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntac cells, SIRC cells, C; II Cells used in the present invention include CHO cells, HEK293 cells, BHK cells, or derivatives thereof), or any other cell type known to those skilled in the art. In some embodiments, the cells used in the present invention are CHO cells, HEK293 cells, BHK cells, or derivatives thereof.

[0143] As used herein, the term "host cell proteins" (HCPs) includes proteins derived from host cells, which may not be related to the desired protein of interest. Host cell proteins may be process-related impurities that may be obtained from the manufacturing process and may include three major categories: cell substrate-derived, cell culture-derived, and downstream-derived. Cell substrate-derived impurities include, but are not limited to, proteins and nucleic acids (host cell genome, vector, or total DNA) derived from the host organism. Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other media components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biochemical processing reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non-metal ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other eluates.

[0144] As used herein, the term "liquid chromatography" refers to a process by which biological / chemical mixtures carried by a liquid can be separated into components as a result of the differential distribution of the components as they flow through a stationary liquid or solid phase. 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 embodiments, a sample containing at least one protein or peptide digest of interest may be subjected to any one or combination of the above-mentioned chromatographic methods. Analytes separated using chromatography will be characterized by distinct retention times that reflect the speed at which the analytes migrate through the chromatography column. Analytes may be compared using chromatograms, which plot retention time on one axis and measured signal on the other, where the measured signal may result, for example, from UV or fluorescence detection.

[0145] As used herein, the term "mass analyzer" includes a device capable of identifying and measuring the exact mass of a specific molecular species. The term is meant to include any molecular detection device capable of characterizing a polypeptide or peptide. A mass analyzer may include three main parts: an ion source, a mass analyzer, and a detection device. The role of the ion source is to generate gas-phase ions. Analyte atoms, molecules, or clusters may be transferred to the gas phase and ionized either simultaneously (as in electrospray ionization) or through a separate process. The choice of ion source depends on the application.

[0146] In some exemplary embodiments, the mass analyzer is a tandem mass analyzer. As used herein, the term "tandem mass analyzer" includes techniques in which structural information about sample molecules is obtained by using multiple stages of mass selection and mass separation. The requirement is that the sample molecules be converted into the gas phase, ionized, and, after an initial mass selection step, fragments are formed in a predictable and controllable manner. Precursor ions are first selected and isolated (MS 1 ), which can be fragmented to obtain meaningful information, 2 Tandem MS has been successfully implemented with a wide variety of analyzer combinations. The choice of analyzer combination for a particular application can be determined by several different factors, such as sensitivity, selectivity, and speed, but also by size, cost, and availability. Two major categories of tandem MS methods are tandem-in-space and tandem-in-time, although there are also hybrids that couple a tandem-in-space analyzer with a tandem-in-space analyzer. A tandem-in-space mass analyzer includes an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. Specific m / z separation functions may be designed so that ions are selected in one section of the instrument, dissociated in an intermediate region, and the product ions are then transmitted to another analyzer for m / z separation and data acquisition. In a tandem-in-time mass analyzer, ions produced in the ion source can be trapped, isolated, fragmented, and m / z separated in the same physical device.

[0147] Peptides identified by mass spectrometry can be used as surrogate representatives of the intact protein and its post-translational or other modifications. They can also be used for protein characterization by correlating experimental and theoretical MS / MS data (the latter generated from possible peptides in protein sequence databases). Characterization includes, but is not limited to, determining the amino acid sequence of a protein fragment, determining protein sequence, determining protein de novo sequence, determining the location of post-translational modifications, or identifying post-translational modifications, or comparability analysis, or a combination thereof.

[0148] In some exemplary embodiments, the mass analyzer may operate with nanoelectrospray or nanospray. The terms "nanoelectrospray" or "nanospray" refer to electrospray ionization at very low solvent flow rates, typically no more than a few hundred nanoliters per minute of sample solution, often without the use of external solvent delivery. The electrospray injection setup that forms the nanoelectrospray may use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs continuous analysis of small sample (analyte) solution volumes over an extended period of time. A dynamic nanoelectrospray emitter uses a capillary column and solvent delivery system to perform chromatographic separation on a mixture before analysis by a mass analyzer.

[0149] In some exemplary embodiments, mass spectrometry can be performed under non-denaturing conditions. As used herein, the term "non-denaturing conditions" can include performing mass spectrometry under conditions that preserve non-covalent interactions in the analyte. For a detailed review of non-denaturing MS, see Review: Elisabetta Boeri Erba & Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE 1176-1192 (2015).

[0150] As used herein, the term "database" refers to a compiled collection of protein sequences that may be present in a sample, for example, in the form of FASTA-formatted files. Suitable protein sequences may be derived from the cDNA sequences of the species under study. Public databases that can be used to search for suitable protein sequences include, for example, databases hosted by Uniprot or Swiss-prot. Databases may also be searched using what are referred to herein as "bioinformatics tools." Bioinformatics tools provide the ability to search uninterpreted MS / MS spectra against all possible sequences in the database(s) and provide 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).

[0151] As used herein, the term "downstream processing technology" refers to one or more technologies used after the upstream processing technology has produced, processed, and / or purified a protein. Downstream processing technologies include, for example, isolation of a protein product using, for example, affinity chromatography, ion exchange chromatography, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, or displacement chromatography.

[0152] 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 & ANDREW L. ZYDNEY, MICROFILTRATION AND ULTRAFILTRATION: PRINCIPLES AND APPLICATIONS (1996), the entire disclosure of which is incorporated herein by reference. Filters with pore sizes smaller than 0.1 μm may be used for ultrafiltration. Using filters with such small pore sizes allows proteins to be retained on the filter, while allowing the permeation of the sample buffer through the filter to reduce the sample volume.

[0153] As used herein, "diafiltration" or "DF" can include methods that use ultrafiltration membranes to remove or exchange salts, sugars, and non-aqueous solvents to separate free of bound species, remove low molecular weight materials, and / or cause changes in the ionic and / or pH environment. Trace solutes are most efficiently removed by adding solvent to the solution being ultrafiltered at a rate approximately equal to the ultrafiltration rate. This washes microspecies from the solution at a constant volume. In certain exemplary embodiments of the present disclosure, a diafiltration step can also be used in connection with the present disclosure to exchange various buffers used, for example, prior to chromatography or other manufacturing steps, as well as to remove impurities from protein preparations.

[0154] As used herein, the term "formulation" refers to a pharmaceutical product, e.g., dupilumab, formulated with one or more pharmaceutically acceptable vehicles.

[0155] As used herein, the term "stable" with respect to protein formulations refers to the ability of the protein of interest within the formulation 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 defined period of time. A formulation may be considered "stable" if, under certain circumstances, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the protein structure or function is maintained after storage for a defined period of time.

[0156] The terms "treat" or "treatment" refer to a therapeutic measure that reverses, stabilizes, or eliminates an undesirable disease or disorder (e.g., atopic dermatitis (eczema), eosinophilic or oral steroid-dependent asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), eosinophilic esophagitis (EoE), inflammation), by causing regression, stabilization, or elimination of one or more symptoms or signs of such a disease or disorder in any clinically measurable way, e.g., with respect to asthma, helping to prevent severe asthma attacks (exacerbations), potentially improving breathing, and helping to reduce the amount of oral corticosteroids needed.

[0157] As used herein, the term "critical quality attribute" (CQA) is used to describe a physical, chemical, biological, or microbiological property or characteristic that must be within appropriate limits, ranges, or distributions to ensure the desired product quality. CQAs can include, for example, post-translational modifications.

[0158] As used herein, "virus filtration" can include filtration using a suitable filter, including, but not limited to, Asahi Kasei Pharma's Planova 20N™, 50N, or BioEx, EMD Millipore's Viresolve™ filters, Sartorius's ViroSart® CPV or Virosart® HF, or Pall Corporation's Ultipor DV20 or DV50™ or Pegasus™ Prime filters. It will be apparent to one skilled in the art to select an appropriate filter to obtain the desired filtration performance.

[0159] Robust and flexible upstream process implementation is desirable for biopharmaceuticals. Efficient upstream processes can lead to desirable production and scale-up of proteins of interest.

[0160] As used herein, a "sample" may be obtained from any step of a bioprocess, such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), any step of downstream processing, drug substance (DS), or drug product (DP), including final formulated product. In certain exemplary embodiments, the sample may be selected from any step of downstream processing of clarification, chromatographic production, or filtration.

[0161] As used herein, the term "protein alkylating agent" or "alkylating agent" refers to an agent used to alkylate specific free amino acid residues in proteins. Non-limiting examples of protein alkylating agents are iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methanemethylthiosulfate (MMTS), and 4-vinylpyridine, or a combination thereof.

[0162] As used herein, "protein denaturation" 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 carried out using a protein denaturant. Non-limiting examples of protein denaturants include heat, high or low pH, and exposure to reducing or chaotropic agents such as DTT and SDS. Several chaotropic agents may be used as protein denaturants. Chaotropic solutes increase the entropy of a system by interfering with intermolecular interactions mediated by non-covalent forces, such as hydrogen bonding, van der Waals forces, and hydrophobic effects. 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-lauroylsarcosine, urea, and salts thereof. In some exemplary embodiments, a denaturant may be used in the mobile phase of 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 can be a surfactant.

[0163] As used herein, the term "digestion" refers to the hydrolysis of one or more peptide bonds of a protein. There are several approaches to performing digestion of proteins in a sample using an appropriate hydrolysis agent, such as enzymatic digestion or non-enzymatic digestion. Digestion of proteins into component peptides can produce "peptide digests" that can be further analyzed using peptide mapping analysis.

[0164] As used herein, the term "hydrolytic agent" refers to any one or combination of a number of different agents that can effect the digestion of proteins. Non-limiting examples of hydrolytic agents capable of enzymatic digestion include protease from Aspergillus saitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin 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) from Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease or biologically active fragments or homologs thereof, or combinations thereof. Non-limiting examples of hydrolysis agents that can perform non-enzymatic digestion include high temperature, microwave, 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 discussing available techniques for protein digestion, see Switzer et al., "Protein Digestion: An Overview of the Available Techniques and Recent Developments" (Linda Switzer, Martin Giera & Wilfried MA Niessen, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 Journal of Proteome Research 1067-1077 (2013)), the entire disclosure of which is incorporated herein by reference. One or a combination of hydrolysis agents can cleave peptide bonds in proteins or polypeptides in a sequence-specific manner to generate a predictable collection of shorter peptides.The ratio of hydrolyzing agent to protein and the time required for digestion can be appropriately selected to obtain optimal digestion of the protein. If the enzyme-to-substrate ratio is inappropriately high, the correspondingly high digestion rate will not allow enough time for peptide analysis by mass spectrometry, resulting in a loss of sequence coverage. On the other hand, a low E / S ratio will require a long digestion time and therefore a long data acquisition time. The enzyme-to-substrate ratio can range from about 1:0.5 to about 1:200.

[0165] As used herein, the term "protein reducing agent" or "reducing agent" refers to an agent used to reduce disulfide bridges in proteins. Non-limiting examples of protein reducing agents used to reduce proteins include dithiothreitol (DTT), β-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or a combination thereof.

[0166] As used herein, the term "protein" or "protein of interest" can include any amino acid polymer having covalently linked amide bonds. Proteins contain one or more amino acid polymer chains, commonly known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues linked through peptide bonds, related naturally occurring structural variants, and synthetic, non-naturally occurring analogs thereof. A "synthetic peptide or polypeptide" refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides may be synthesized, for example, using an automated polypeptide synthesizer. A variety of solid-phase peptide synthesis methods are known to those skilled in the art. A protein can comprise one or multiple polypeptides that form a single functional biomolecule. In another exemplary embodiment, proteins can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, etc. Proteins of interest can include biotherapeutic proteins, recombinant proteins used in research or therapy, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, and human antibodies. 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), may also be used to produce the proteins.For a recent review discussing biotherapeutic proteins and their production, see, e.g., 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 disclosure of which is incorporated herein by reference). In some exemplary embodiments, the proteins comprise modifications, adducts, and other covalently linked moieties. These modifications, adducts, and moieties include, for example, avidin, streptavidin, biotin, glycans (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 may be classified based on composition and solubility, and thus can include simple proteins, e.g., globular proteins and fibrous proteins; conjugated proteins, e.g., nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, e.g., primary and secondary derived proteins.

[0167] As used herein, the term "recombinant protein" refers to a protein produced as a result of 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, humanized, or 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 the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0168] The term "antibody," as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and 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 are further divided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the present disclosure, the FRs of an anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs. The term "antibody," as used herein, also includes antigen-binding fragments of full-length antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, 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 obtained from full-length antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or may be synthesized.The DNA may be sequenced and manipulated chemically or by using molecular biology techniques to place one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0169] As used herein, "antibody fragment" includes portions of an intact antibody, such as the antigen-binding or variable region of the antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining regions (CDRs), as well as triabodies, tetrabodies, 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 variable regions of immunoglobulin light and heavy chains are linked by a peptide linker. In some exemplary embodiments, an antibody fragment contains sufficient amino acid sequence of the parent antibody from which it is a fragment to bind 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 can be produced by any means. For example, antibody fragments may be enzymatically or chemically produced by fragmentation of an intact antibody and / or may be recombinantly produced from a gene encoding a partial antibody sequence. Alternatively, or in addition, antibody fragments may be wholly or partially synthetically produced. Antibody fragments may optionally comprise single-chain antibody fragments. Alternatively, or in addition, antibody fragments may comprise multiple chains linked together, for example, by disulfide linkages. Antibody fragments may optionally comprise multimolecular complexes. Functional antibody fragments typically comprise at least about 50 amino acids, more typically at least about 200 amino acids.

[0170] The term "monoclonal antibody," as used herein, is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies may be obtained by any means available or known in the art from a single clone, including eukaryotic, prokaryotic, or phage clones. A wide variety of techniques known in the art may be used to prepare monoclonal antibodies useful in the present disclosure, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0171] As used herein, a "protein pharmaceutical product," "biopharmaceutical product," or "biotherapeutic agent" includes an active ingredient that may be wholly or partially biological in nature. In one embodiment, a protein pharmaceutical product may include a peptide, a protein, an antibody, an antigen, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, a cell, a tissue, or a combination thereof. In another embodiment, a protein pharmaceutical product may include a recombinant, engineered, modified, mutated, or truncated form of a peptide, a protein, an antibody, an antigen, a vaccine, a peptide-drug conjugate, a protein-drug conjugate, a cell, a tissue, or a combination thereof. Exemplary Anti-IL-4Rα Antibodies

[0172] As used herein, dupilumab (IgG4 isotype) is a covalent heterotetramer consisting of two disulfide-linked human heavy chains, each covalently linked through a disulfide bond to a human kappa light chain. Each heavy chain contains a serine to proline mutation at amino acid 233, located in the hinge region of the Fc domain, reducing the tendency of the antibody to form half-antibodies in solution. Each heavy chain contains a single N-linked glycosylation site (Asn), located within the CH2 domain of the Fc constant region of the molecule. 302 Antibodies based on the primary sequence (no N-linked glycosylation) are composed of 16 disulfide bonds and a Lys from the C-terminus of each heavy chain. 452Taking into account the removal of α, it has a molecular weight of 146,897.0 Da. The heavy and light chain variable domains combine to form the complementarity-determining regions (CDRs) through which dupilumab binds to its target, interleukin-4 receptor alpha (IL-4Rα). A schematic diagram of dupilumab, including the location of N-linked glycosylation sites and disulfide bond structures, is presented in Figure 1.

[0173] A manufacturing process may be used to produce a human antibody or antigen-binding fragment thereof that specifically binds to 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 the amino acid sequence of SEQ ID NO: 1. The antibody or antigen-binding fragment may comprise three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) having the 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 may be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that may be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a combination of 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 are also available for identifying CDR sequences within antibodies.

[0174] In certain embodiments, the antibody or antigen-binding fragment thereof comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) from the heavy and light chain variable region amino acid sequence pair (HCVR / LCVR) of SEQ ID NOs: 1 and 2.

[0175] In certain embodiments, the antibody or antigen-binding fragment thereof comprises six CDRs (HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3) having the amino acid sequences of SEQ ID NOs: 3 / 4 / 5 / 6 / 7 / 8.

[0176] 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.

[0177] In a specific embodiment, the antibody is dupilumab, which comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 1 and 2.

[0178] In a specific embodiment, the antibody is dupilumab comprising the heavy chain / light chain amino acid sequence pair of SEQ ID NOs: 9 and 10. Dupilumab HCVR amino acid sequence:

[0179] EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVSSISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRLSITIRPRYYGLDVWGQGTTVTVS (SEQ ID NO: 1). Dupilumab LCVR amino acid sequence:

[0180] DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTPYTFGQGTKLEIK (SEQ ID NO: 2). Dupilumab HCDR1 amino acid sequence:

[0181] GFTFRDYA (SEQ ID NO: 3). Dupilumab HCDR2 amino acid sequence:

[0182] ISGSGGNT (SEQ ID NO: 4). Dupilumab HCDR3 amino acid sequence:

[0183] AKDRLSITIRPRYYGL (SEQ ID NO: 5). Dupilumab LCDR1 amino acid sequence:

[0184] QSLLYSIGYNY (SEQ ID NO: 6). Dupilumab LCDR2 amino acid sequence:

[0185] LGS (SEQ ID NO: 7). Dupilumab LCDR3 amino acid sequence:

[0186] MQALQTPYT (sequence number 8). Dupilumab HC amino acid sequence:

[0187] EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVSSISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRLSITIRPRYYGLDVWGQ GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 9) (amino acids 1-124 = HCVR; amino acids 125-451 = HC constant). Dupilumab LC amino acid sequence:

[0188] DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10) (amino acids 1-112 = LCVR; amino acids 112-219 = LC constant).

[0189] In the specific embodiment, the manufacturing method is used: 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-43 / SCB-VH-92 VL-44 / SCB-VH-92;SCB-VL-44 / SCB-VH-62;SCB-VL-44 / SCB-VH-68;SCB-VL-44 / SCB-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;SCB-VL-45 / SCB-VH-92;SCB-VL-46 / SCB-VH-92;SCB-VL-47 / SCB-VH-92;SCB-VL-48 / SCB-VH-92;SCB-VL-49 / SCB-VH-92 VH-92;SCB-VL-50 / SCB-VH-92;SCB-VL-51 / SCB-VH-92;SCB-VL-51 / SCB-VH-93;SCB-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-VL-54 / SCB-VH-68;SCB-VL-54 / SCB-VH-72;SCB-VL-54 / SCB-VH-82;SCB-VL-54 / SCB-VH-82 VL-54 / SCB-VH-85;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;SCB-VL-55 / SCB-VH-85;SCB-VL-55 / SCB-VH-91;SCB-VL-56 / SCB-VH-92;SCB-VL-57 / SCB-VH-92;SCB-VL-57 / SCB-VH-93;SCB-VL-57 / SCB-VH-92 VH-59;SCB-VL-57 / SCB-VH-60;SCB-VL-57 / SCB-VH-61;SCB-VL-57 / SCB-VH-62;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;SC B-VL-57 / SCB-VH-70;SCB-VL-57 / SCB-VH-71;SCB-VL-57 / SCB-VH-72;SCB-VL-57 / SCB-VH-73;SCB-V L-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 / SC B-VH-86;SCB-VL-57 / SCB-VH-87;SCB-VL-57 / SCB-VH-88;SCB-VL-57 / SCB-VH-89;SCB-VL-57 / SCB-V The antibody or antigen-binding fragment thereof may be produced comprising a light chain variable region (LCVR) and heavy chain variable region (HCVR) sequence pair (LVCR / HCVR) selected from the group consisting of H-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;

[0190] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the LCVR / HCVR sequence pair of SCB-VL-44 / SCB-VH-92.

[0191] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the LCVR / HCVR sequence pair of SCB-VL-54 / SCB-VH-9.

[0192] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the LCVR / HCVR sequence pair of SCB-VL-55 / SCB-VH-92.

[0193] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an 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 an LCVR comprising the LCDR1 sequence of SCB-55-LCDR1, the LCDR2 sequence of SCB-55-LCDR2, and the LCDR3 sequence of SCB-55-LCDR3.

[0194] 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.

[0195] 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.

[0196] The antibodies listed in Table 1 below are described in more detail in US Pat. No. 10,774,141, which is incorporated by reference in its entirety for all purposes. Table 1 [Table 1] TIFF2025507792000002.tif222165TIFF2025507792000003.tif223165TIFF2025507792000004.tif227165

[0197] In certain embodiments, the manufacturing method may be used to produce an antibody or antigen-binding fragment thereof comprising a light chain variable region (LCVR) and 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.

[0198] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the LCVR / HCVR pair MEDI-37GL-VL / MEDI-37GL-VH.

[0199] 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.

[0200] The antibodies listed in Table 2 below are described in more detail in US Pat. No. 8,877,189, which is incorporated by reference in its entirety for all purposes. Table 2 [Table 2] TIFF2025507792000006.tif219166TIFF2025507792000007.tif219166TIFF2025507792000008.tif21716 6TIFF2025507792000009.tif217166TIFF2025507792000010.tif219166TIFF2025507792000011.tif61166

[0201] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the LCVR / HCVR pair of AJOU-90-VL / AJOU-83-HV.

[0202] 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.

[0203] 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 by reference in their entirety for all purposes. Table 3 [Table 3] TIFF2025507792000013.tif219166TIFF2025507792000014.tif217166

[0204] In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises 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.

[0205] The antibodies listed in Table 4 below are described in more detail in US Pat. No. 7,605,237 and US Pat. No. 7,608,693, which are incorporated by reference in their entirety for all purposes. Table 4 [Table 4] TIFF2025507792000016.tif119166

[0206] The present disclosure is not limited to any of the aforementioned protein(s), protein(s) of interest, antibody(ies), cell culture medium, host cell protein(s), protein alkylating agent(s), protein denaturing agent(s), protein reducing agent(s), digestive enzyme(s), treatment(s), process(es), sample(s), surfactant(s), detergent(s), chromatography method(s), filtration method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH, temperature(s), or concentration(s), and may include any suitable It is understood that the means may select any protein(s), protein(s) of interest, antibody(ies), cell culture medium, host cell protein(s), protein alkylating agent(s), protein denaturant(s), protein reducing agent(s), digestive enzyme(s), treatment(s), process(es), sample(s), surfactant(s), detergent(s), chromatography method(s), filtration method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH, temperature(s), or concentration(s).

[0207] The present disclosure will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the invention. Cell Culture Media and Supplements

[0208] Removing serum and reducing or eliminating hydrolysates from cell culture media reduces lot-to-lot variability and enhances downstream processing steps, but unfortunately reduces cell growth, viability, and protein expression. Therefore, chemically defined serum-free and low- or no-hydrolysate media require additional components to improve cell growth and protein production. The cell culture media of the present invention may be supplemented with additional components 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 requirements of the cells to be cultured or the desired cell culture parameters.

[0209] In some embodiments, the cell culture media disclosed herein are supplemented with polyamines, such as ornithine, putrescine, spermine, spermidine, or a combination ("PS medium"), to improve cell growth, cell viability, and recombinant protein production.

[0210] In one embodiment, the PS medium comprises 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, 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, 619, 620, 625, 630, 635, 640, 645, 650, 700, 750, 800, 850, or 900 ± 14 µM (expressed in micromoles per liter). In one embodiment, PS medium contains ornithine at a concentration between about 0.09 and 0.9 mM.

[0211] In one embodiment, and in addition to the inclusion of ornithine or putrescine, the medium comprises a nucleoside mixture 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.

[0212] In one embodiment, the medium contains about 174 μM±26 μM nucleosides. In one embodiment, the medium contains a purine derivative at 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 about 105 μM. In one embodiment, the medium contains about 106 μM±5 μM purine derivatives. Purine derivatives include hypoxanthine, and nucleosides, adenosine, and guanosine. In one embodiment, the medium contains a pyrimidine derivative at 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. In one embodiment, the medium contains 68 μM±5 μM of pyrimidine derivatives. Pyrimidine derivatives include nucleosides, thymidine, uridine, and cytidine. In one specific embodiment, the medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

[0213] In addition to the inclusion of ornithine or putrescine, in one embodiment, the medium also contains an amino acid at a cumulative concentration of at least 40 mM, where the amount of glutamine is not included in the cumulative total calculation. In one embodiment, glutamine is not included in the medium, but may be provided to the medium as a "point-of-use additive" during cell culture, e.g., protein production. Thus, in some embodiments, for example, in methods of culturing cells or producing a protein of interest, the medium may be supplemented with glutamine as a point-of-use additive. 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 glutamine-supplemented medium is 2 mM±0.5 mM.

[0214] In one embodiment, in addition to the inclusion of ornithine or a combination of ornithine and putrescine, the medium also contains amino acids having nonpolar side chain groups at a concentration of at least 15 mM, at least 24 mM, at least 25 mM, at least 26 mM, at least 27 mM, at least 28 mM, at least 29 mM, or at least 30 mM. In one embodiment, the medium contains about 30 mM of amino acids having nonpolar side chain groups. In one embodiment, 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% of the total amount of amino acids by mole contained in the medium are amino acids having nonpolar side chain groups. In one embodiment, about 42%±1% of the amino acids by mole in the medium are amino acids having nonpolar side chains. Amino acids having nonpolar side chain groups include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. In one embodiment, the optional mixture of amino acid supplements is selected from the group consisting of the amino acids in Table 5 below. Table 5 [Table 5]

[0215] In one embodiment, in addition to the inclusion of ornithine or a combination of both ornithine and putrescine, the medium also contains an amino acid having an uncharged polar side chain group at a concentration of about 10 mM to 34 mM, about 11 mM to 33 mM, about 12 mM to 32 mM, about 13 mM to 31 mM, about 14 mM to 30 mM, about 15 mM to 29 mM, about 16 mM to 28 mM, about 17 mM to 27 mM, about 18 mM to 26 mM, about 19 mM to 25 mM, about 20 mM to 24 mM, about 21 mM to 23 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM. In one embodiment, the medium contains about 22 mM of an amino acid having an uncharged polar side chain group. In another embodiment, the medium contains about 12 mM of an amino acid having an uncharged polar side chain group. In one embodiment, 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% of the total molar amount of amino acids contained in the medium are amino acids having uncharged polar side chain groups. In one embodiment, about 30% ± 3% of the amino acids in the medium are amino acids having uncharged polar side chain groups. Amino acids having uncharged polar side chains include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0216] In one embodiment, in addition to the inclusion of ornithine or a combination of both ornithine and putrescine, the medium also contains an amino acid (e.g., an acidic amino acid) that is negatively charged at pH 6 at a concentration of about 4 mM to 14 mM, about 5 mM to 13 mM, about 6 mM to 12 mM, about 7 mM to 11 mM, about 8 mM to 10 mM, about 9 mM, or about 4 mM. In one embodiment, the medium contains about 9 mM of an acidic amino acid. In one embodiment, the medium contains 9 mM ± 1 mM of an acidic amino acid. In one embodiment, about 8% to 18%, about 9% to 17%, about 10% to 16%, about 11% to 15%, about 12% to 14%, or about 13% of the total amount of amino acids by mole contained in the medium are acidic amino acids. In one embodiment, about 12.6% ± 1% of the amino acids by mole in the medium are acidic amino acids. Acidic amino acids include aspartic acid and glutamic acid.

[0217] In one embodiment, in addition to the inclusion of ornithine or a combination of both ornithine and putrescine, the medium also contains an amino acid (e.g., a basic amino acid) that is positively charged at pH 6 at 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. In one embodiment, the medium contains about 11 mM basic amino acids. In one embodiment, the medium contains about 11.42 mM±1 mM basic amino acids. In one embodiment, 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% of the total amount of amino acids by mole contained in the medium are basic amino acids. In one embodiment, about 16% of the amino acids by mole in the medium are basic amino acids. In one embodiment, about 15.8%±2.4% of the amino acids in the medium are basic amino acids, and in one embodiment, about 21%±3.2% of the amino acids in the medium are basic amino acids, including lysine, arginine, and histidine.

[0218] In one embodiment, in addition to the inclusion of ornithine or a combination of both ornithine and putrescine, the medium also contains 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, about 42% by mole of the amino acids in the medium are nonpolar amino acids, about 30% by mole are uncharged polar amino acids, about 13% by mole are acidic amino acids, and about 16% by mole are basic amino acids.

[0219] In one embodiment, in addition to the inclusion of ornithine or a combination of both ornithine and putrescine, the medium contains micromolar amounts of fatty acids (or fatty acid derivatives) and tocopherol. In one embodiment, the fatty acids include any one or more of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid. In one embodiment, the medium contains tocopherol, linolenic acid, and thioctic acid.

[0220] In one embodiment, the medium also includes a vitamin mixture, which includes other nutrients and essential nutrients at a cumulative concentration of at least about 700 μM or at least about 2 mM. In one embodiment, the vitamin mixture includes one or more of D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemi-Ca), riboflavin, thiamine HCl, vitamin B12, etc. In one embodiment, the vitamin mixture includes D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemi-Ca), riboflavin, thiamine HCl, and vitamin B12.

[0221] In one embodiment, the medium also contains taurine, hypotaurine, or a combination thereof.Taurine is present in many tissues of humans and other mammalian species, such as the brain, retina, cardiac muscle, skeletal muscle, smooth muscle, platelets, and neutrophils.Taurine can assist in osmoregulation, membrane stabilization, and anti-inflammation, and also regulates mitochondrial protein synthesis through enhancing the activity of the electron transport chain, which protects against superoxide generation (Jong et al., 2010, Journal of Biomedical Science 17(Suppl 1):S25; Jong et al., 2012, Amino Acids 42:2223-2232sw). In one embodiment, the serum-free cell culture medium can contain about 0.1 mM to about 10 mM taurine, about 0.1 mM to about 1 mM taurine, about 0.4 mM to about 8 mM taurine, about 1 mM to about 6 mM taurine, about 2 mM to about 5 mM taurine, or about 3 mM to about 4 mM taurine. Taurine has also been found to increase cell-specific productivity, allowing these cells to produce less ammonia byproducts, improving recombinant protein production. See U.S. Pat. No. 10,927,342, incorporated herein by reference in its entirety.

[0222] In one embodiment, a fed-batch production method for making dupilumab includes culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding the dupilumab at 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.

[0223] In one embodiment, the dupilumab is produced at a titer of at least 1.5 g / L in the cell culture production medium by day 4. In another embodiment, the dupilumab is produced at a titer of at least 2 g / L in the cell culture production medium by day 4.

[0224] In one embodiment, the culturing continues for about 10 to 18 days, hi another embodiment, the culturing continues for about 10 days, about 12 days, about 14 days, about 16 days, or about 18 days.

[0225] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium comprises recombinant growth factors, an osmolality regulator, a pH buffer, glutamine, and a cytoprotectant.

[0226] In one embodiment, the CHO cells are cultured at a temperature ranging from about 32°C to about 38°C.

[0227] In one embodiment, the dupilumab is produced at a titer of at least about 0.5 g / L in the cell culture production medium on day 4. In another embodiment, the dupilumab is produced in the cell culture production medium such that the cell viability 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.

[0228] In one embodiment, a fed-batch production method for making dupilumab includes culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding the dupilumab at a large scale in a cell culture production medium, wherein tyrosine in the medium is supplemented on day 3 at a concentration of about 2 g / L.

[0229] In one embodiment, the dupilumab is produced at a titer of at least about 8 g / L in the cell culture production medium on day 14.

[0230] In one embodiment, the dupilumab is produced at a titer of at least 9 g / L in the cell culture production medium on days 10 to 14. In another embodiment, the dupilumab is produced at a titer of at least 10 g / L in the cell culture production medium on days 10 to 14.

[0231] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium comprises recombinant growth factors, an osmolality regulator, a pH buffer, glutamine, methotrexate, and a cytoprotectant.

[0232] In one embodiment, the CHO cells are cultured at a temperature ranging from about 32°C to about 38°C.

[0233] In one embodiment, the ammonia concentration in the cell culture production medium on day 14 is less than about 10 mM. In another embodiment, the ammonia concentration in the cell culture production medium on day 14 is less than about 8 mM.

[0234] In one embodiment, a fed-batch production method for making dupilumab includes culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding the dupilumab at a large scale in a cell culture production medium, wherein tyrosine in the medium is supplemented on days 3 and 7 at a concentration of about 2 g / L.

[0235] In one embodiment, a fed-batch production method for making dupilumab includes culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding the dupilumab at a large scale in a cell culture production medium, wherein tyrosine in the medium is supplemented on days 3 and 7 at a concentration of about 1 g / L.

[0236] In one embodiment, the dupilumab is produced at a titer of at least about 8 g / L in the cell culture production medium on day 14. In another embodiment, the dupilumab is produced at a titer of at least 9 g / L in the cell culture production medium. In yet another embodiment, the dupilumab is produced at a titer of at least 10 g / L in the cell culture production medium.

[0237] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium comprises recombinant growth factors, an osmolality regulator, a pH buffer, glutamine, methotrexate, and a cytoprotectant.

[0238] In one embodiment, the CHO cells are cultured at a temperature ranging from about 32°C to about 38°C.

[0239] In one embodiment, a fed-batch production method for making dupilumab includes culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding the dupilumab at a large scale in a cell culture production medium, wherein sodium phosphate in the medium is supplemented on days 2 and 4, or on days 0, 2, 4, 6, and / or 8, at a concentration of about 200-550, about 200-275, about 250-325, about 300-375, about 350-425, about 400-475, about 450-525, or about 500-550 mg / L, such that the titer of the dupilumab in the cell culture production medium is about 5 g / L, about 6 g / L, about 7 g / L, or about 8 g / L on day 10. In another embodiment, the titer of dupilumab in the cell culture production medium 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 on days 10 to 14.

[0240] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium comprises recombinant growth factors, an osmolality regulator, a pH buffer, glutamine, methotrexate, and a cytoprotectant.

[0241] In one embodiment, the CHO cells are cultured at a temperature ranging from about 32°C to about 38°C.

[0242] In one embodiment, the sodium phosphate in the medium is supplemented on days 2 and 4, or on days 0, 2, 4, 6, and / or 8, at a concentration of about 200-550, about 200-275, about 250-325, about 300-375, about 350-425, about 400-475, or about 450-525 mg / L.

[0243] In one embodiment, a fed-batch production method for making dupilumab includes culturing, on a large scale, Chinese hamster ovary (CHO) cells comprising a nucleic acid encoding dupilumab in a cell culture production medium, wherein sodium phosphate in the medium is supplemented on days 2 and 4, or on days 0, 2, 4, 6, and 8, at a concentration of about 200-550, about 200-275, about 250-325, about 300-375, about 350-425, about 400-475, about 450-525, or about 500-550 mg / L, respectively; tyrosine in the medium is supplemented on day 3 at a concentration of about 2 g / L; and insulin in the medium is supplemented on days 2 and 4 at a concentration of about 7.5 mg / L, such that the titer of dupilumab in the cell culture production medium is at least 5 g / L on day 10.

[0244] In one embodiment, a fed-batch production method for making dupilumab includes culturing, on a large scale, Chinese hamster ovary (CHO) cells comprising a nucleic acid encoding dupilumab in a cell culture production medium, wherein sodium phosphate in the medium is supplemented on days 2 and 4, or on days 0, 2, 4, 6, and 8, at a concentration of about 200-550, about 200-275, about 250-325, about 300-375, about 350-425, about 400-475, about 450-525, or about 500-550 mg / L, respectively; tyrosine in the medium is supplemented on days 3 and 7 at a concentration of about 1 g / L; and insulin in the medium is supplemented on days 2 and 4 at a concentration of about 7.5 mg / L, such that the titer of dupilumab in the cell culture production medium is at least 5 g / L on day 10.

[0245] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium comprises recombinant growth factors, an osmolality regulator, a pH buffer, glutamine, methotrexate, and a cytoprotectant.

[0246] In one embodiment, the CHO cells are cultured at a temperature ranging from about 32°C to about 38°C.

[0247] In one embodiment, the sodium phosphate in the medium is replenished at a concentration of about 200-550, about 200-275, about 250-325, about 300-375, about 350-425, about 400-475, or about 450-525 mg / L on days 2 and 4, or on days 0, 2, 4, 6, and 8. In one embodiment, the culture continues for about 10 days.

[0248] Various embodiments of the media of the present invention include combinations of any of the above embodiments, including chemically defined, hydrolysate-free, serum-free media containing ornithine or putrescine in the amounts indicated, and additionally containing, among other things, (a) amino acids; (b) optionally nucleosides; (c) salts of divalent cations; (d) fatty acids and tocopherols; and (e) vitamins. In some embodiments, small amounts of hydrolysates may be added to the PS medium.

[0249] Applicants contemplate that any one or more or combinations of a variety of base media supplemented with ornithine or a combination of both ornithine and putrescine may be used in the practice of the present invention. Base media are generally known in the art and include, inter alia, Eagle's MEME (Minimum Essential Medium) (Eagle, Science, 1955, 112(3168):501-504), Ham's F12 (Ham, Proc. Nat'l. Acad. Sci. USA, 1965, 53:288-293), F-12 K medium, Dulbecco's medium, Dulbecco's modified Eagle's medium (Proc. Natl. Acad. Sci. USA., 1952 August; 38(8):747-752), DMEM / Ham's F12 1:1, Trowell's T8, A2 medium (Holmes and Wolf, Biophys. Biochem. Cytol., 1961, 10:389-401), Waymouth's medium (Davidson and Waymouth, Biochem. J., 1945, 39(2):188-199), Williams E medium (William's et al., Exp. Cell Res., 1971, 69:105 et seq.), RPMI 1640 (Moore et al., J. Amer. Med. Assoc., 1967, 199:519-524), MCDB 104 / 110 medium (Bettger et al., Proc. Nat'l. Acad. Sci. USA, 1981, 78(9):5588-5592), Ventrex HL-1 medium, albumin-globulin medium (Orr et al., Appl. Microbiol., 1973, 25(1):49-54), RPMI-1640 medium, RPMI-1641 medium, Iscove's modified Dulbecco's medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media such as EX-CELL™ 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 4,072,565), 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 medium permutations (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.).

[0250] In certain embodiments, the medium is chemically defined and contains, in addition to ornithine or a combination of both ornithine and putrescine: CaCl2 2H2O; HEPES buffer, KCL; MgSO4; NaCl; Na2HPO4 or other phosphate salts; pyruvate; L-alanine; L-arginine HCl; L-asparagine HCl HO; L-aspartic acid; L-cysteine ​​HCl HO; L-glutamic acid; glycine; L-histidine HCl HO; L-isoleucine; L-leucine; L-lysine HCl; L-methionine; L-ornithine HCl; L-phenylalanine; L-proline; L-serine; L-threonine; L-tryptophan; L-tyrosine 2Na 2 H2O; L-valine; D-biotin; choline chloride; folic acid; myo-inositol; niacinamide; pyridoxine HCl; D-pantothenic acid; riboflavin; thiamine HCl; vitamin B12; p-benzoic acid; ethanolamine HCl; poloxamer 188; DL-α-tocopheryl phosphate; linoleic acid; Na2SeO3; thioctic acid; and glucose; and optionally adenosine; guanosine; cytidine; uridine; thymidine; and hypoxanthine disodium.

[0251] In one embodiment, the starting osmolality of the medium is 200-500, 250-400, 275-350, or about 300 mOsm. During growth of the cells in the medium, and after any feeding, particularly according to a fed-batch protocol, the osmolality of the culture can increase up to as much as about 350, 400, 450, or 500 mOsm.

[0252] In some embodiments where the osmolality of the defined medium is less than about 300, the osmolality is brought to about 300 by adding a specified amount of excess one or more salts. In one embodiment, the osmolality is increased to a desired level by adding one or more osmolytes selected from sodium chloride, potassium chloride, magnesium salts, calcium salts, amino acid salts, fatty acid salts, sodium bicarbonate, sodium carbonate, potassium carbonate, chelator salts, sugars (e.g., galactose, glucose, sucrose, fructose, etc.), and combinations thereof. In one embodiment, the osmolytes are added in addition to the concentrations of components already present in the defined medium (e.g., sugars are added in addition to the concentrations specified for the sugar component).

[0253] The present disclosure provides a cell culture comprising a cell line expressing a protein of interest in OS medium as described above. In one embodiment, the cell culture comprises insulin, which may be added to the medium as a point-of-use component or may be included in the 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, among others. 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.

[0254] Animal cells, e.g., CHO cells, can be cultured in small-scale cultures, e.g., 125 mL vessels containing about 25 mL of medium, 250 mL vessels containing about 50-100 mL of medium, or 500 mL vessels containing about 100-200 mL of medium. Alternatively, cultures can be cultured on a larger scale, e.g., 1000 mL vessels containing about 300-1000 mL of medium, 3000 mL vessels containing about 500-3000 mL of medium, 8000 mL vessels containing about 2000-8000 mL of medium, and 15000 mL vessels containing about 4000-15000 mL of medium. Manufacturing cultures can contain large-scale cultures, e.g., 10,000 L or more of medium. Large-scale cell cultures, e.g., those for clinical production of protein therapeutics, are typically maintained for several days or even weeks, during which time the cells produce the desired protein(s). During this period, the culture may be supplemented with a concentrated feed medium containing components consumed during the course of the culture, e.g., nutrients and amino acids. Concentrated feed media can be based on any cell culture medium formulation. Such concentrated feed media can contain most of the components of a cell culture medium, for example, about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or even about 1000X of their normal useful amounts. Concentrated feed media are often used in fed-batch culture processes.

[0255] In some embodiments, cell culture media are supplemented with "point-of-use additives," also known as additives, point-of-use components, or point-of-use chemicals, during the course of cell growth or protein production. Point-of-use additives include any one or more of 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 proliferation or, in some cases, cell differentiation. Growth factors are typically proteins (e.g., insulin), small peptides, or steroid hormones, such as estrogen, DHEA, and testosterone. In some cases, growth factors may be non-naturally occurring chemicals that promote cell growth or protein production, such as tetrahydrofolate (THF), methotrexate, and the like. Non-limiting examples of protein and peptide growth factors include angiopoietin, bone morphogenetic proteins (BMPs), 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), migration stimulating factor, These include 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-alpha (TNF-α), vascular endothelial growth factor (VEGF), Wnt signaling pathway agonists, placental growth factor (PlGF), fetal bovine somatotrophin (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 point-of-use additives, the growth factor insulin.In one embodiment, the insulin concentration in the medium, e.g., the amount of insulin in the cell culture medium after addition, is about 0.1 μM to 10 μM. One or more point-of-use additives may be included in the medium formulation of some embodiments.

[0256] Buffers are generally known in the art. The present invention is not limited to any particular buffer or buffers, and any person skilled in the art may select an appropriate buffer or buffer system for use with a particular cell line producing a particular protein. In one embodiment, the ready-to-use additive buffer is NaHCO3. In one embodiment, the ready-to-use additive buffer comprises NaHCO3. In another embodiment, the buffer is HEPES.

[0257] Energy sources for use as point-of-use additives during cell culture are also well known in the art. Without limitation, in one embodiment, the point-of-use additive energy source is glucose. Taking into account the specific requirements of a particular cell line and the protein to be produced, in one embodiment, glucose may be added to a cumulative concentration of about 1 to 35 mM in the medium. In some cases, glucose may be added at a high level of up to 10 g / L.

[0258] Chelators are also well known in the art of cell culture and protein production. Tetrasodium EDTA dihydrate and citrate are two common chelators used in the art, but other chelators may be used in the practice of the present invention. In one embodiment, the chelator added at the time of use is tetrasodium EDTA dihydrate. In one embodiment, the chelator added at the time of use is citrate, for example, Na3C6H5O7.

[0259] In one embodiment, the cell culture may be supplemented with one or more point-of-use added amino acids, such as glutamine. In one embodiment, the cell culture medium is supplemented with point-of-use added glutamine at a final concentration of about 1 mM to 13 mM.

[0260] Other point-of-use additives include various amino acids and metal salts, such as one or more of iron, nickel, zinc, and copper salts. In one embodiment, the cell culture medium is supplemented with any one or more of copper sulfate, zinc sulfate, iron chloride, and nickel sulfate.

[0261] In one embodiment, the cell culture medium is supplemented with any one or more of the following point-of-use additives: about 25-30 mM NaHCO3, about 1.5-2 mM glutamine, about 0.75-0.9 μM insulin, about 10.75-11.25 mM glucose, about 6.25-6.75 μM zinc sulfate, about 0.15-0.17 μM copper sulfate, about 70-80 μM iron chloride, about 0.6-0.7 μM nickel sulfate, about 80-90 μM EDTA, and about 45-60 μM citrate.

[0262] In one embodiment, the medium is replenished at intervals during cell culture according to a fed-batch process, which is commonly known in the art and used for optimized protein production (see YM Huang et al., Biotechnol Prog. 2010 Sep-Oct;26(5):1400-10).

[0263] Cell viability, viable cell density, and cell doublings are improved compared to cells grown in culture without ornithine or putrescine. With respect to cell viability, cells grown in PS medium exhibit 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-fold greater viability than similar or identical cells grown in non-PS medium.

[0264] In some embodiments, the doubling rate of surviving 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 higher than the doubling rate of mammalian cells grown in non-PS medium. In some embodiments, the doubling rate of surviving mammalian cells in PS medium is 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.

[0265] In some embodiments, the doubling time of actively cycling 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 cycling mammalian cells in PS medium is about 24±1 hours. In some embodiments, the doubling time of actively dividing mammalian 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 mammalian cells cultured in non-PS medium. Protein production

[0266] In addition to chemically defined PS medium and methods for culturing cells in PS medium, the present disclosure provides methods for producing proteins, such as therapeutically effective antibodies or other biopharmaceutical drug substances, in cells cultured in PS medium.

[0267] In some embodiments, the rate of protein production by mammalian cells cultured in PS medium is at least 5%, 10%, 15%, or 20% higher than the rate of protein production by the same mammalian cells cultured in non-PS medium. In some embodiments, the rate of protein production 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.

[0268] In some embodiments, the protein production yield or titer, which may be expressed in 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.

[0269] 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 diabody, a triabody, or a tetrabody, 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.

[0270] In some embodiments, the protein of interest is a recombinant antibody comprising an Fc portion and another domain, hi some embodiments, the Fc portion comprises the hinge region followed by the CH2 and CH3 domains of IgG.

[0271] The present invention is not limited to any particular type of cell 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 cells may be stem cells or recombinant cells transformed with a vector for recombinant gene expression, or cells transfected with a virus to produce a viral product. The cells may contain a recombinant heterologous polynucleotide construct encoding a protein of interest. This construct may be episomal or may be a physically integrated element within the cellular genome. The cells may also produce a protein of interest without the protein encoded on a heterologous polypeptide construct. In other words, the cells may naturally encode the protein of interest, such as antibody-producing B cells. The cells may also be primary cells, such as chicken embryo cells, or primary cell lines. Examples of useful 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 DUX B-11, Veggie-CHO, GS-CHO, S-CHO, or a CHO lec mutant line.

[0272] In one embodiment, the cell, which is a CHO cell, ectopically expresses a 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 a human or rodent immunoglobulin CH2 and CH3 region. In one embodiment, the protein comprises a human or rodent immunoglobulin CH1, CH2, and CH3 region. 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).

[0273] The production phase can be performed at any scale of culture, from flasks or wave bags to 1-liter bioreactors and large-scale industrial bioreactors. Large-scale processes can be performed at volumes ranging from about 1,000 liters to 25,000 liters or more. Protein production can be controlled using one or more of several means, such as temperature shift 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 about 30°C to 36°C, or about 30°C to 34°C. Additionally, chemical inducers of protein production, such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylene bisacetamide (HMBA), can be added simultaneously with, before, or after the temperature shift. If the inducer is added after the temperature shift, it may be added 1 hour to 5 days after the temperature shift, for example, 1 to 2 days after the temperature shift. The production cell culture may be run as a continuous-feed culture system, such as in a chemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-Dec; 17(6):1032-41), or according to a fed-batch process (Huang, 2010).

[0274] The present invention is useful for improving protein production through cell culture processes. The cell lines used in the present invention can be genetically engineered to express polypeptides of commercial or scientific interest. Genetic engineering of cell lines involves transforming or transducing cells with recombinant polynucleotide molecules or modifying host cells to express a desired recombinant polypeptide (e.g., by homologous recombination and gene activation or fusion of recombinant and non-recombinant cells). Methods and vectors for genetically engineering cells or cell lines to express a polypeptide of interest are well known to those skilled in the art; for example, various techniques are exemplified in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and revised quarterly); 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 for the production of complex recombinant proteins such as cytokines, clotting 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).The dihydrofolate reductase (DHFR)-deficient mutant cell line (Urlaub et al. (1980), Proc Natl Acad Sci USA 77: 4216-4220) DXBI1 is a desirable CHO host cell because efficient DHFR selection and an amplifiable gene expression system allow high-level recombinant protein expression in these cells (Kaufman RJ. (1990), Meth Enzymol 185:537-566). Furthermore, these cells can be easily manipulated as adherent or suspension cultures and exhibit relatively good genetic stability. CHO cells and the proteins recombinantly expressed by them have been extensively characterized and approved for use in commercial manufacturing. In some embodiments, the CHO cell line is described in U.S. Patent Application Nos. 2010 / 0304436 A1, 2009 / 0162901 A1, and 2009 / 0137416 A1, and U.S. Patent Nos. 7,455,988 B2, 7,435,553 B2, and 7,105,348 B2.

[0275] The present disclosure is not limited in scope by the specific embodiments described herein, which are intended as illustrations of individual aspects or embodiments of the invention. Functionally equivalent methods and components are within the scope of the invention. Various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the invention.

[0276] 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, viability, and polypeptide production from recombinantly engineered animal cell lines (or native cells) expressing a protein of interest, thereby increasing culture robustness and improving yield of the polypeptide of interest. Seed Train Optimization and Measurement

[0277] As described herein, an improved method for optimizing large-scale protein production in cell culture has been developed by implementing a seed train process that biases cells to increase viable cell density (VCD) in the large-scale production vessel. It was previously observed in U.S. application Ser. No. 16 / 984,581, entitled "Metabolically Optimized Cell Culture," incorporated herein by reference, that peak lactate consumption during the seed train can be used to improve large-scale protein production. As described herein, a novel seed train process has been developed that further enhances cell metabolism and productivity by adjusting the initial VCD, demonstrating a novel, distinct mechanism for enhancing protein production from that described in U.S. application Ser. No. 16 / 984,581, without affecting the observed lower peak lactate consumption.

[0278] By way of background, it is well known that protein production through cell culture is customarily carried out using a batch or fed-batch process. The initial stage of inoculum growth after vial thawing involves culturing cells in a seed culture. Culture vessels include, but are not limited to, well plates, wave bags, T-flasks, shake flasks, stirred vessels, spinner flasks, hollow fiber, airlift bioreactors, and the like. A suitable cell culture vessel is a bioreactor. A bioreactor refers to any culture vessel manufactured or engineered to manipulate or regulate environmental conditions. Such culture vessels are well known in the art. Typically, cells, such as in a seed train bioreactor, are grown at an exponential growth rate to progressively increase the size and / or volume of the cell population. After the cell mass is scaled up through several bioreactor stages, the cells are then transferred to a production bioreactor while still in exponential growth (log phase) (Gambhir, A. et al., 2003, J Bioscience Bioeng 95(4):317-327). Allowing cells in batch cultures, such as seed cultures, to go through the log phase and into stationary phase is generally considered undesirable. It has been recommended that cultures be passaged while in the log phase, among other reasons, before cells, such as adherent cells, reach confluence due to contact inhibition or before the accumulation of waste products inhibits cell growth (Cell Culture Basics, Gibco / Invitrogen Online Handbook, www.invitrogen.com; ATCC® Animal Cell Culture Guide, www.atcc.org).

[0279] After transfer to fed-batch culture, the cells are cultured for a period of time while monitoring and adjusting the medium composition to allow production of the protein or polypeptide of interest. After reaching a specific yield or determining that the culture should be terminated due to cell viability, waste accumulation, or nutrient depletion, the produced protein or polypeptide is isolated. Efforts have been made to improve the total final protein titer, particularly those related to reducing metabolic waste product output during cell culture, such as the accumulation of lactate. These efforts have focused on controlling glucose- or nutrient-limited fed-batch processes (see, e.g., WO2004104186; U.S. Pat. No. 8,192,951B2), improving cell culture medium conditions (e.g., U.S. Pat. No. 7,390,660; Zagari, et al., 2013, New Biotechnol., 30(2):238-45), or cellular 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).

[0280] Controlled feeding of cells has been used to achieve more efficient metabolic phenotypes (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 is complicated by the fact that nutrient depletion 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 approach is to grow cells in fed-batch to moderately high densities and then deliberately induce an extended productive stationary phase, for example, by temperature or pH shift (Quek et al., 2010, Metab Eng 12(2):161-71. doi: 10.1016 / j.ymben.2009.09.002. Epub 2009 Oct. 13).

[0281] In some embodiments, the initial VCD during the seed train process is adjusted to enhance protein production during a batch or fed-batch process. In some embodiments, the initial VCD is adjusted to 1.7x10 at each step (N-5 to N-1). 5 ~2.3x10 5 Compared to the acceptable standard initial VCD range of cells / mL, approximately 3.5x10 5 ~5.43x10 5 In some embodiments, the initial VCD is about 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, or 3.0x larger than another initial VCD in an acceptable standard seed train.

[0282] In some embodiments, increasing the initial VCD in the N-5 to N-1 vessels does not result in a change in the final VCD of N-1 compared to a standard seed train. In one aspect, the optimized seed train results in an increase in the final VCD in the N-5 to N-2 vessels compared to a standard seed train. In one aspect, there is no substantial difference in the peak lactic acid observed in the final production vessel compared to a standard seed train. In one aspect, the maintenance biomass of the optimized seed train is increased. In one aspect, the optimized seed train results in a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% increase in final titer (g / L).

[0283] Example 22 is provided to describe to one of ordinary skill in the art how to use an optimized seed train and is not intended to limit the scope of what the inventors regard as the invention.

[0284] Due to the importance of VCD in seed train processes and in-process control, more efficient and improved methods for measuring VCD are needed. Currently, assays used to monitor VCD in suspension mammalian cell line cell cultures are based on offline analysis (Ritacco, Biotechnology Progress, 34(6):1407-1426, 2018). For example, the trypan blue exclusion test (TBET) can visually distinguish viable from nonviable cells using a manual hemocytometer or automated bioanalyzer; however, offline assays are not compatible with the Food and Drug Administration (FDA) regulatory framework for implementing mechanisms to design, analyze, and control pharmaceutical manufacturing processes through inline or online measurement of critical process parameters (CPPs) that affect critical quality attributes (CQAs) (e.g., Process Analytical Technology / PAT) (Metze, Bioprocess and Biosystems Engineering, 43(2):193-205, 2020). Offline assays also introduce delays between sampling and response to process changes, requiring more labor and resources.

[0285] It will be appreciated that a need exists for in-line and / or online methods and systems for optimizing cell culture therapeutic protein production and measuring VCD that are consistent with the regulatory framework outlined by the FDA for process analytical technology practices.

[0286] Dielectric permittivity measurement is a promising technology for real-time analysis of cell culture and biomass properties. Dielectric permittivity measurement is a non-destructive, non-invasive, continuous process monitoring tool that can generate continuous process data for both suspension and adherent cell cultures. Nevertheless, the implementation of biomass online monitoring techniques remains challenging due to the associated complex calibration process, integration difficulties, and poor model accuracy and transferability (Carvell, Cytotechnology, 50:35-48, 2006; Kell et al., J. Bioelectricity, 4(2):317-348, 1990). Therefore, the present application provides the benefits of a permittivity measurement system and method that can accurately determine viable cell density predictions online during cell culture.

[0287] In one embodiment, the present disclosure provides a method for culturing cells by measuring a first property of the cell culture, predicting a second property of the cell culture using the first property, and adjusting culture conditions based on the predicted second property of the cell culture.

[0288] In one embodiment, a capacitance probe can be a capacitance or dielectric sensor that generates a change in a dielectric field and measures the dielectric constant of the surrounding medium. A capacitance probe can use at least one electrode to generate the change in an electric field. The electrodes in a capacitance probe can include platinum, aluminum, gold, or other suitable metals.

[0289] In one embodiment, an online sensor is used to measure a first property of the cell culture.

[0290] In one embodiment, a correlation equation is used to predict a second property of the cell culture based on a measured first property of the cell culture.

[0291] In one embodiment, the cell culture property can be a capacitance value, a viable cell density, a dielectric constant, and / or a conductivity.

[0292] In one embodiment, the present disclosure provides a method for measuring the VCD of cells cultured in a bioreactor by applying an electric field to the cells cultured in the bioreactor, measuring the capacitance, and correlating the measured capacitance to viable cell density.

[0293] In one embodiment, the present disclosure provides a method for culturing cells by measuring a first property of the cell culture using an online sensor, predicting at least one measurement of a second property of the cell culture using the measurement of the first property of the cell culture and a correlation equation, and adjusting culture conditions to culture the cells based on the predicted measurement of the at least one second property of the cell culture.

[0294] In one embodiment, an offline assay is used to measure the second property of the first cell culture.

[0295] In one embodiment, the cells are from the same cell line as the cell line used to generate the correlation equation.

[0296] In one embodiment, the cells are from a different cell line than the cell line used to generate the correlation equation.

[0297] In one embodiment, the correlation equation is obtained using more than one cell line.

[0298] In one embodiment, more than one first capacitance value of the first cell culture is measured.

[0299] In one embodiment, more than one first VCD value of the first cell culture is determined.

[0300] In one embodiment, at least 50 percent of the variability in the first VCD value is due to variation in the first capacitance value.

[0301] In one embodiment, multivariate data analysis is used to generate correlation equations.

[0302] In one embodiment, bioanalyzer measurements of cell culture samples can be performed using a bioanalyzer system. The bioanalyzer system can include at least one 96-well plate, syringe, 24-position external sample tray, lab-on-a-chip, or a combination thereof. A bioanalyzer system for performing bioanalyzer measurements of cell culture samples can be automated. The bioanalyzer system can analyze a single sample by simultaneously running about 1 to about 20 assays. Exemplary, non-limiting bioanalyzer measurements that can be performed by the bioanalyzer system include quantification of pH, CO₂ partial pressure (pCO₂), O₂ partial pressure, glucose, lactate, glutamine, glutamate, ammonia, sodium, potassium, calcium, osmolality, total cell density, viable cell density, cell viability, or a combination thereof. The bioanalyzer system can determine viable cell density using a trypan blue exclusion test. Cell culture samples in a lab-on-a-chip can be analyzed using at least one electrophoretic separation, flow cytometry, or a combination thereof. Electrophoretic separation can be used to perform DNA, RNA, or protein assays, or a combination thereof. A DNA assay can determine the size or quantity of DNA, or the size and quantity of DNA in a cell culture sample. An RNA assay 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. A protein assay can determine the size or quantity of at least one protein in a sample. Flow cytometry can be used to analyze protein expression, apoptosis, gene silencing transfection monitoring, intracellular staining, extracellular staining, or a combination thereof.

[0303] In one embodiment, the cell culture nutrients can include, for example, glucose, glutamine, glutamic acid, sodium, potassium, calcium, O2, or a combination thereof.

[0304] In one embodiment, cell culture metabolites can include, for example, acetic acid, acetate, lactic acid, lactate, ammonia, ethanol, lactic acid, CO2, or combinations thereof.

[0305] 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 dielectric constant of the surrounding medium, a standard viability assay including trypan blue exclusion, or a combination thereof. Cell density refers to the number of cells in a given volume of culture medium.

[0306] Example 23 is provided to illustrate to one of ordinary skill in the art how to use the improved capacitance probe to measure VCD and is not intended to limit the scope of what the inventors regard as their invention. Vessel and bioreactor agitation, aeration and measurement optimization

[0307] During the growth and production phases, optimization of starting volumes, air sparging, and agitation set points in vessels and bioreactors is important because cell cultures are exposed to shear stress from agitator impellers and sparged gassing, which is necessary to maintain sufficient oxygen transport and dissolved carbon dioxide uptake to maintain cell health. It has been found that these processes can be optimized by staging or varying agitation and air sparging rates. For example, staging or varying agitation and air sparging rates has been found to provide greater improvements in VCD. Dissolved oxygen and carbon dioxide can be controlled to set points through sparging in a cascade regulatory loop.

[0308] It has also been found that heterogeneity can be reduced by lowering the amount of acidic and basic species in the recombinant protein of interest through stepwise or varying agitation and aeration rates. These variations can result from deamidation, sialylation, glycosylation, and fragmentation, which can alter the stability, activity, and strength of proteins containing Fc portions (portions derived from antibody crystallizable region fragments). Sissolak et al., J. Indust. Microbiol. Biotech. 46: 1167-78 (2019). Basic mutations can increase antibody binding to Fc receptors. Hintersteiner et al., MABS 8: 1458-60 (2016).

[0309] Fc glycans may play a role in immunogenicity, bioactivity, pharmacokinetics, and pharmacodynamics. Reusch and Tejada, Glycobiol. 25: 1325-34 (2015). A possible phenomenon is known as non-glycosylated heavy chain (NGHC). NGHC variation can alter effector functions, such as opsonization. Opsonization involves the Fc portion, which is involved in ADCC (antibody-dependent cellular cytotoxicity), 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 Fc portions.

[0310] In one exemplary embodiment, the agitation of the culture may be increased by 25%, 50%, 75%, 100%, 125%, 150%, 175%, or 200% on days 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11. In one embodiment, the initial agitation rate is 20-150 rpm, increased to 40-300 rpm, and then increased again to 80-600 rpm. Agitation rpm may also be correlated to force per unit volume using known models. For example, for a 10,000 L bioreactor with two impellers rotating at 40 rpm, the power per unit volume is equal to 0.076 hp / 1000 L; for a 7,500 L bioreactor rotating at 22 rpm, the power per unit volume is equal to 0.017 hp / 1000 L. For a 3,000 L bioreactor with two impellers rotating at 40 rpm, the power per unit volume is equal to 0.033 hp / 1000 L; for a 2,500 L bioreactor rotating at 40 rpm, the power per unit volume is equal to 0.04 hp / 1000 L. For a 10,000 L bioreactor with a single impeller rotating at 40 rpm, the power per unit volume is equal to 0.005 hp / 1000 L; for a 7,500 L bioreactor rotating at 22 rpm, the power per unit volume is equal to 0.001 hp / 1000 L.

[0311] In another embodiment, the initial stirring speed is about 75 rpm, then increased to 150 rpm, and then increased to 225 rpm. In one exemplary embodiment, the aeration 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, 1 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 day, or 11 day. In one embodiment, the initial aeration rate is between 50 and 75 slpm, increasing to about 225 to 275 slpm on day 2. In one embodiment, the sparger has 146 to 292 holes ranging in size from 0.5 mm to 2 mm, and may include spargers with 146x0.5 mm holes, 146x1.0 mm holes, 146x1.5 mm holes, 146x2.0 mm holes, 292x0.5 mm holes, 292x1.0 mm holes, 292x1.5 mm holes, and 292x2.0 mm holes.

[0312] In one embodiment, the agitation rate and air sparging rate are increased on the same day. In another embodiment, the agitation rate and air sparging rate are increased on different days, in which case the agitation rate can be increased on the feed day, as shown in Figure 45.

[0313] In one exemplary embodiment, the starting volume of the vessel or bioreactor is optimized to reduce agitation-related surface effects. The initial working volume in the vessel or bioreactor is typically set at a level with sufficient empty space to provide headroom for volume adjustment, including subsequent feed. Unexpectedly, it has been found that increasing the initial working volume increases biomass and final titer. It has been found that increasing the working volume reduces agitation-related surface effects caused by the impeller in the bioreactor, including the air cascade effect, and provides greater control over dissolved oxygen concentration.

[0314] In one embodiment, the initial working volume of the vessel or bioreactor is adjusted so that the top impeller (if more than one) is covered by cell culture medium at the start of the culturing process. Shown below in Table 6 are examples of optimized volumes (kg) for different vessels and to reduce agitation-related surface effects. Table 6 [Table 6]

[0315] 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 measured automatically at set times. In one aspect, the aeration rate can be set to automatically adjust based on the measured dissolved oxygen level using cascade control to maintain a set level of dissolved oxygen, which can be constant, variable, or stepped as described above.

[0316] As mentioned above, dissolved oxygen and carbon dioxide concentrations are critical process parameters that must be carefully controlled and require optimized systems and methods for measuring dissolved gases. In one embodiment, an improved probe for measuring dissolved gases in large-scale vessels and bioreactors used to culture cells in the production of therapeutic protein products in the biopharmaceutical industry is disclosed. An improved data processing method was developed through the comparison of data from two optical probes (designated Optical Probe A and Optical Probe B) with electrochemical probe data in a large-scale industrial bioreactor. As shown in Examples 19 and 21 below, a calibration method was developed that takes into account the offset observed between both optical probes (Optical Probe A: ∼+1.6% saturation, Optical Probe B: ∼−3.0% saturation) relative to the electrochemical probe, with the goal of obtaining the required accuracy while minimizing maintenance and reducing or eliminating undesired excursion events outside the normal operating range. In particular, the optical probe demonstrated 100% elimination of excursions above the normal operating range observed on the electrochemical probe and approximately a two-fold reduction in noise compared to the electrochemical probe (based on a standard deviation comparison).

[0317] Additionally, improved signal processing methods involving data filtering and smoothing have been developed for electrochemical probes to reduce deviation events determined to be false positives, as shown in Example 20. In one embodiment, signal processing is applied in real time.

[0318] In one embodiment, cells are cultured in a large-scale vessel or bioreactor where dissolved gas levels, including oxygen, are measured periodically. In one aspect, the time 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.

[0319] In one embodiment, a vessel or bioreactor with reduced maintenance requirements is configured to use one or more optical probes that measure dissolved oxygen. Bioreactors and vessels may also be configured with one or more optical probes that measure other gases, such as carbon dioxide. In one embodiment, data from the optical probes is processed based on a fixed offset. In one embodiment, the offset is based on a correlation of data from the optical probe and the electrochemical probe. In another embodiment, the offset is based on a correlation of the optical probe and a known standard. In another embodiment, data from the optical probes is processed based on an offset that varies over time during the culture process. In one embodiment, the offset is applied in real time.

[0320] 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 embodiment, the sampling window for the data is smoothed between a 10 minute window and a 33 minute window.

[0321] It is believed that the same methods for improving accuracy and reducing signal noise used with optical and electrochemical probes to measure dissolved oxygen can also be applied in measuring carbon dioxide and other dissolved gases.

[0322] In one embodiment, cells are cultured under appropriate pCO2, as more fully described in U.S. Patent Application No. 63 / 246,047 (Methods of Controlling Antibody Heterogeneity), which allows for control of heterogeneity of recombinantly produced antibodies, as well as derivatives and fragments, in mammalian cells. The cells can be any suitable mammalian cell, including CHO, BHK, HEK293, HeLa, human amniotic fluid, Per.C6, and Sp2 / 0 cells. Without being bound by any theory, it is believed that increasing CO2 levels in the medium increases intracellular CO2, which may be involved in charge and glycosylation variations. This effect is separate from any decrease in pH, presumably due to carboxylic acid formation.

[0323] Carbon dioxide concentration can be increased using CO2 sparging or by reducing air sparging. Reducing the pressure in the production bioreactor reduces oxygen solubility; this in turn requires more oxygen sparging to maintain the dissolved oxygen (DO) set point and increase the gas flow rate, which drives pCO2 from the culture medium. Carbon dioxide concentration can be measured using a CO2 electrode, also called a Severinghaus electrode. More advanced systems, such as the BioProfile® FLEX and FLEX2 Analyzers, are commercially available. Charge variants can be measured using imaging capillary isoelectric focusing (iCIEF) and ion-exchange chromatography with salt gradient elution. NGHC can be measured by reduced capillary electrophoresis (CE)-SDS.

[0324] The cells may be cultured for about 10 to 15 days. In one embodiment, the cells may be cultured for about 14 days. The pCO2 conditions may be about 30 mmHg to about 210 mmHg, 50 mmHg to 200 mmHg, 60 mmHg to 190 mmHg, 70 mmHg to 180 mmHg, 80 mmHg to 170 mmHg, 90 mmHg to 160 mmHg, 100 mmHg to 150 mmHg, 110 mmHg to 140 mmHg, 120 mmHg to 140 mmHg, or any value within this range during culture, and this is preferably maintained by CO2 sparging and may be measured using a CO2 electrode.

[0325] 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 the IL-4 receptor; and culturing the cells under pCO2 conditions that allow the mammalian cells to produce the Fc-containing protein capable of binding to the IL-4 receptor. Preferably, the protein is a human monoclonal antibody, preferably an IgG antibody including subclasses such as IgG1 and IgG4.

[0326] In another embodiment, the Fc-containing protein produced by the cells, e.g., the main peak form of an antibody capable of binding to the IL-4 receptor, can comprise about 38% to about 65% of the total Fc-containing protein; the Fc-containing protein, e.g., an acidic variant of an antibody capable of binding to the IL-4 receptor, can comprise about 20% to about 47% of the total Fc-containing protein; and the Fc-containing protein, e.g., an antibody capable of binding to a basic variant of the IL-4 receptor, can comprise up to about 36% of the total Fc-containing protein. The percentage of Fc-containing protein comprising a nonglycosylated heavy chain can include about 3% to about 8%, 4% to 7%, 5% to 7%, 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or decimal value within these ranges.

[0327] In another embodiment, the major peak form of the Fc-containing protein produced by the cells, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 50% to about 70% of the total Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, total antibody, antibody derivative, or antibody fragment, acidic variants of the Fc-containing protein can comprise about 20% to about 47%, and basic variants of the Fc-containing protein can comprise up to about 15%. The proportion of Fc-containing protein comprising a non-glycosylated heavy chain, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 3% to about 8%, 4% to 7%, 5% to 7%, 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or decimal value within these ranges.

[0328] In another embodiment, the major peak form of the Fc-containing protein produced by the cells, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 50% to about 70% of the total Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, total antibody, antibody derivative, or antibody fragment; the acidic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 20% to about 47%; and the basic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise up to about 6%, about 8%, or about 10%. The proportion of the Fc-containing protein comprising a non-glycosylated heavy chain, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 3% to about 8%, 4% to 7%, 5% to 7%, 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or decimal value within these ranges.

[0329] In another embodiment, the major peak form of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, produced by the cells can comprise about 50% to about 65% of the total Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, total antibody, antibody derivative, or antibody fragment; the acidic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 23% to about 46%; and the basic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise up to about 15%. The proportion of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, comprising a non-glycosylated heavy chain can include about 3% to about 8%, 4% to 7%, 5% to 7%, 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or decimal value within these ranges.

[0330] In another embodiment, the major peak form of the Fc-containing protein produced by the cells, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 50% to about 65% of the total Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, total antibody, antibody derivative, or antibody fragment; the acidic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 31% to about 46%; and the basic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise up to about 15%. The proportion of the Fc-containing protein comprising a non-glycosylated heavy chain, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 3% to about 8%, 4% to 7%, 5% to 7%, 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or decimal value within these ranges.

[0331] In another embodiment, the major peak form of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, produced by the cells can comprise about 50% to about 65% of the total Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, total antibody, antibody derivative, or antibody fragment; the acidic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 23% to about 39%; and the basic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise up to about 15%. The proportion of Fc-containing protein comprising a non-glycosylated heavy chain, e.g., an antibody capable of binding to IL-4 receptor, can include about 3% to about 8%, 4% to 7%, 5% to 7%, 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or decimal value within these ranges.

[0332] In another embodiment, the major peak form of the Fc-containing protein produced by the cells, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 35% to about 60% of the total Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, total antibody, antibody derivative, or antibody fragment; the acidic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 20% to about 40%; and the basic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 10% to about 40%. The proportion of Fc-containing protein comprising a non-glycosylated heavy chain, e.g., an antibody capable of binding to IL-4 receptor, can include about 3% to about 8%, 4% to 7%, 5% to 7%, 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or decimal value within these ranges.

[0333] In another embodiment, the major peak form of the Fc-containing protein produced by the cells, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 39% to about 50% of the total Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, total antibody, antibody derivative, or antibody fragment; an acidic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 22% to about 38%; and a basic variant of the Fc-containing protein, e.g., an antibody capable of binding to IL-4 receptor, can comprise about 14% to about 36%. The proportion of Fc-containing protein comprising a non-glycosylated heavy chain, e.g., an antibody capable of binding to IL-4 receptor, can include about 3% to about 8%, 4% to 7%, 5% to 7%, 5% to 6.5%, 5% to 6%, 5% to 5.75%, 5% to 5.5%, or any integer or decimal value within these ranges.

[0334] Further details are further described in Example 18 below, based on an Fc-containing protein, e.g., an antibody capable of binding to the IL-4 receptor, which is illustrative of various embodiments of the invention, but does not limit the invention in any way, and which, with respect to dupilumab, may be applied to cells cultured under appropriate pCO2 conditions to regulate acidic species and non-glycosylated heavy chains.

[0335] In one exemplary embodiment, the glucose feeding strategy is also optimized to prevent hyperosmolality conditions. It has been found that glucose concentration affects osmolality conditions, which can affect cell culture performance. To improve cell culture conditions and performance, the lower dextrose target was reduced to prevent hyperosmolality conditions. In one embodiment, the dextrose target was reduced by 10%, 20%, 30%, 40%, 50%, 60%, or 70%. In one embodiment, the dextrose target was increased stepwise on days 2, 3, 4, 5, or 6. In one embodiment, the dextrose target was increased stepwise after 0.5, 1, 1.5, 2, 2.5, or 3 days, and then decreased. In one exemplary embodiment, the initial dextrose target is between 5 g / L and 7 g / L, and the incremental dextrose target is between 7 g / L and 11 g / L. In one embodiment, the dextrose target is incrementally increased and decreased one or more times. Exemplary dextrose targets and adjustments over time are shown in Figures 58A and 58B.

[0336] In another embodiment, high temperature, short time (HTST) treatment of seed train and production media and feeds at 102°C for 10 seconds may be performed. In another embodiment, HTST treatment is carried out for 8, 9, 11, 12, 13, 14, or 15 seconds at temperatures of about 101°C, 102°C, 103°C, 104°C, 105°C, or about 106°C. HTST can function as a flash pasteurization of cell culture media and feeds and can be used as a viral barrier for upstream processing during production, mitigating the risk of adventitious pathogens and resulting in comparable process performance and product quality, including titer. Centrifugation, depth and polish filtration

[0337] In certain exemplary embodiments, primary 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 associated cellular debris. Shown in Figure 28 is an exemplary schematic diagram of the use of depth, polish, and guard filters.

[0338] Centrifugation of the sample can be performed, for example, but not by way of limitation, at 7,000 x g to approximately 12,750 x g. A 60-80% solids filling of the bowl is recommended for calculating the discharge interval. The concentrate is then processed by in-line depth, polish, and guard filtration. The resulting filter flux is a function of the centrifuge feed flow rate and the area of ​​the depth, polish, and guard filters. In some embodiments, a bowl speed of 5550 rpm (7000 g) and a feed flow rate of approximately 1630 (L / h) can be used. The filter flux and discharge interval can be calculated as shown in Table 7 below. Table 7 [Table 7]

[0339] It has been found that bowl speed can affect turbidity by altering the settling velocity for particles of various sizes. In a large-scale production context, such centrifugation can occur online with flow rate settings to achieve, for example, turbidity levels of 150 NTU in the resulting supernatant.

[0340] To obtain the desired impurity removal and product recovery from the depth filtration step, the cell culture medium or centrate may need to be adjusted to a specific pH and conductivity.

[0341] To reduce lot-to-lot variability, centrate turbidity should be measured at the midpoint between discharges, known as the bowl midpoint (t=½ discharge interval), to determine if adjustments to bowl 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 result in lower turbidity clearance on the filter. Due to the altered levels of centrifugation-induced shear and therefore particle size distribution with altered bowl speed, centrifuge performance has been found to be impossible to predict by the use of linear extrapolation using the sigma concept alone.

[0342] To overcome these limitations, a predictive profiler was created to visualize factors and responses as reflected in Figure 29.

[0343] The resulting turbidity model was significant (p<0.0001) with an R2adj of 0.95 and an RMSE of 25.0 FNU. The main and quadratic effects of relative centrifugal force, the main effect of flow rate, and the interaction of relative centrifugal force and flow rate were identified as important factors. Flow rate had the largest effect on centrate turbidity. Generally, at RCFs below 8000 g, centrate turbidity was solely a function of flow rate.

[0344] The centrate was then collected for further processing or filtered in-line through one or more depth filters for further sample clarification. Depth filters, most frequently used in bioprocessing, are composed of diatomaceous earth, cellulose fibers, and a positively charged resin binder. Unlike membrane filters, depth filters retain particles throughout the porous filter medium, allowing for the retention of both particles larger and smaller than the pore size. Particle retention is thought to involve both size exclusion and adsorption through ionic, hydrophobic, and other mechanisms. Filter fouling mechanisms can include pore blocking, cake formation, and pore narrowing. Non-limiting examples of depth filters that can be used in the context of this disclosure include Millistak+ X0HC, F0HC, D0HC, C0HC, A1HC, and B1HC depth filters (EMD Millipore), 3M™ Models 30 / 60ZA, 60 / 90ZA, VR05, and VR07, and delipidized depth filters (3M Corp.). A 0.2 μm filter, such as a Sartorius 0.45 / 0.2 μm Sartopore™ bilayer or a Millipore Express SHR or SHC filter, typically follows the depth filter. Emphaze AEX Hybrid Purifier multifunction filters can also be used to filter the depth filtration material. Other filters known to those skilled in the art can also be used.

[0345] After depth filtration, polish filtration is used to ensure final solids removal before column chromatography. Polish filtration may include specially designed filters that increase impurity absorption through an ionic mechanism, thus reducing the overall impurity level applied to the primary column chromatography step. Guard filtration after polish filtration is used solely for bioload control.

[0346] To provide high titer and increased protein production, the filter pore size for these polish and guard filters may range from 0.15 μm to 0.25 μm, and in one embodiment, may be 0.2 μm. For an average 10,000 L bioreactor culture, the polish filter loading is 255 L / m 2 ~270L / m 2 In one embodiment, the range is about 265 to 270 L / m 2 (For example, 33.6m per 10,000L batch 2 ) The guard filter loading may be 700 L / m 2 ~720Lm 2 may be in the range of Protein A Chromatography

[0347] In certain exemplary embodiments, it may be suitable to subject a biological sample to affinity chromatography for the production of a protein of interest. The chromatography material is capable of selectively or specifically binding to or interacting with the protein of interest. Non-limiting examples of such chromatography materials include: Protein A and Protein G. Also included are, for example, chromatography materials comprising a protein or portion thereof capable of binding to or interacting with the protein of interest.

[0348] Affinity chromatography can involve subjecting a biological sample to a column containing a suitable Protein A resin. As used herein, the term "Protein A" refers to Protein A recovered from its natural source, Protein A produced synthetically (e.g., by peptide synthesis or by recombinant technology), and Protein A. H 2 / C HProtein A resins include those variants that retain the ability to bind to proteins having the Fc region. Protein A can additionally bind to human IgG molecules containing the IgG F(ab')2 fragment from the human VH3 gene family (Roben et al., J Immunol. 1995 154(12):6437-45). In certain embodiments, Protein A resins, if they possess the Fc partial region, are useful for affinity-based production and isolation of diverse antibody isotypes by specifically interacting with that portion of the molecule.

[0349] 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 SuRe pcc, MabSelect Xtra, rProtein A Sepharose from Cytiva, ProSep HC, ProSep Ultra, ProSep Ultra Plus from EMD Millipore, MabCapture from ThermoFisher, and Amsphere™ A3 from JSR Life Sciences.

[0350] Prior to sample loading, the affinity column may be equilibrated with an appropriate buffer. The pH of Protein A loading may be, for example, about 6 to about 8, about 6 to about 7, about 7 to about 8, or about 6. After loading the column, the column may be washed one or more times using an appropriate wash buffer. The column may then be eluted using an appropriate elution buffer, such as glycine-HCl, acetic acid, or citric acid. The eluate may be monitored using techniques well known to those skilled in the art, such as a UV detector. The eluted fractions of interest may be collected and then prepared for further processing.

[0351] Protein A wash buffers may be selected based on their ability to disrupt protein-protein interactions, such as interactions between the protein of interest and impurities such as HCPs, without disrupting interactions between the protein of interest and the chromatographic material. Suitable wash buffers for HCP removal may contain, for example, salts (e.g., sodium-containing salts, e.g., sodium phosphate; potassium-containing salts, e.g., potassium sorbate; magnesium-containing salts; hydrochloric acid-containing salts, e.g., guanidine hydrochloride; Tris-containing salts), detergents (e.g., polysorbate 20, polysorbate 80), polar materials (e.g., isopropanol, ethanol), or amino acids (e.g., arginine). Suitable wash buffers for HCP removal may have a pH of about 5 to about 9, such as about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9.

[0352] An appropriate wash buffer can be selected based on removal of a particular HCP of concern for the manufacturing process, for example, by selecting a wash buffer pH that corresponds to the pI of the HCP of concern. The wash buffer can be selected, for example, based on disrupting electrostatic interactions between the HCP of concern and the protein of interest, hydrophobic interactions between the HCP of concern and the protein of interest, or both. The specific properties of the HCP of concern can be used to inform the selection of buffer components, for example, as shown in Table 8. HCP profiling can be used to identify HCPs of concern by identifying and quantifying HCPs that associate with the protein of interest, for example, using mass spectrometry. An appropriate wash buffer can be selected based on ensuring acceptable yield of the protein of interest by minimizing binding of the protein of interest to the chromatography resin as much as possible. Table 8. Exemplary buffer components and pH for disrupting protein-protein interactions during affinity capture [Table 8]

[0353] Affinity wash buffers can further be selected based on their ability to remove HMW species of the protein of interest. An appropriate wash buffer can be selected that results in a substantial reduction in HMW species while maintaining an acceptable yield of the protein of interest.

[0354] Reduction of HCP levels and / or HMW species using affinity wash buffers may allow for achieving adequate drug substance quality with fewer downstream chromatography steps, for example, by using a Protein A column and an AEX column without a CEX or HIC column; or by using a Protein A column followed by a mixed-mode column and an AEX column without a CEX or HIC column. If the impurity levels achievable with additional columns cannot be matched solely through affinity capture wash buffer optimization, the use of charged filter media during the filtration step may have an additional contribution. This is typically performed after the viral inactivation step, during which some pH adjustment has already been made, making targeting specific conditions for filtration easily integrated into downstream processes. Filter media with multiple mechanisms of removal, including cationic, anionic, hydrophobic, and mixed-mode mechanisms, are commercially available. Operating the filter under conditions in which the desired product does not bind increases the potential for impurity removal and high recovery, as the charge density on the filter is typically lower compared to packed-bed chromatography. Low pH retention

[0355] In certain exemplary embodiments, the primary recovery process may also reduce or inactivate viruses that may be present in the biological sample. Any one or more of a variety of methods of virus reduction / inactivation may be used during the primary recovery phase of production, including heat inactivation (pasteurization), pH inactivation, buffer / detergent treatment, UV and gamma irradiation, and the addition of certain chemical inactivators, such as β-propiolactone, or copper phenanthroline, as described in U.S. Pat. No. 4,534,972, the entire disclosure of which is incorporated herein by reference. In another embodiment, virus-retentive filtration may also be used as a specialized virus clearance step, as discussed below.

[0356] In exemplary embodiments where viral reduction / inactivation is used, in one aspect, the biological sample may be adjusted, if necessary, for further production steps. For example, after low pH viral inactivation, the pH of the sample may be adjusted to a more neutral pH, e.g., about 4.5 to about 8.5, before continuing the production process. In another aspect, the mixture may also be diluted with water for injection (WFI) to obtain the desired conductivity.

[0357] In another embodiment, the eluate may be subjected to viral inactivation, for example, by either detergent or low pH. An appropriate detergent concentration or pH (and time) may be selected to obtain the desired viral inactivation result.

[0358] In another embodiment, after viral inactivation, the eluate can be pH and / or conductivity adjusted for subsequent production steps, including anion exchange chromatography, cation exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., Protein A), and size exclusion chromatography, among others. Anion Exchange Chromatography

[0359] In certain exemplary embodiments, a biological sample is subjected to at least one anion exchange (AEX) separation step to produce a protein of interest. In one scenario, the anion exchange step can occur after an affinity chromatography method (e.g., Protein A affinity). In another scenario, the anion exchange step can occur before an affinity chromatography step. In some embodiments, the AEX separation is the second of four chromatography unit operations and occurs downstream of viral inactivation by affinity capture and low pH hold operations. In yet other embodiments, the AEX separation precedes an ion exchange step. Alternatively, the AEX separation follows another ion exchange method.

[0360] Anion exchange packed bed chromatography is based on ionic interactions between binding entities (target proteins or impurities) and functional groups immobilized on the chromatographic medium. Performance can be a function of the mobile phase, functional groups, and resin backbone. In some embodiments, the specific purpose of the AEX step is to reduce the levels of DNA, HCPs, HMW species, and viruses, if present.

[0361] 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 chromatographic methods, such as affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, as well as other modes of chromatography known to those skilled in the art.

[0362] In carrying out the separation, the initial protein composition (biological sample) may be contacted with the anion exchange material using any of a variety of techniques, for example, by using batch production techniques or chromatographic techniques.

[0363] In the context of batch production, the anion exchange material is prepared in or equilibrated with a desired starting buffer. During preparation, a slurry of the anion exchange material may be obtained. The biological sample may be contacted with the slurry to allow protein adsorption to the anion exchange material. The solution containing acidic species that do not bind to the AEX material may be separated from the slurry by allowing the slurry to settle and removing the supernatant. The slurry may be subjected to one or more washing and / or elution steps.

[0364] In the context of chromatographic separations, packed-bed chromatography columns are used to contain a chromatographic support material (resin or solid phase). A sample containing the protein of interest is loaded onto the specific chromatography column. The column may then be subjected to one or more washing steps using an appropriate wash buffer. Components of the sample that are not adsorbed onto the resin will pass through the column. Components that are adsorbed to the resin can be differentially eluted using an appropriate elution buffer.

[0365] 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 120 g / L, about 15 ... 00g / L, approximately 105g / L, approximately 110g / L, approximately 115g / L, approximately 120g / L, approximately 125g / L, approximately 130g / L, approximately 135g / L, approximately 140g / L, approximately 145g / L, approximately 150g / L, about 155 g / L, about 160 g / L, about 165 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.

[0366] In some embodiments, the sample may be neutralized before contacting with the AEX material. The sample, e.g., a viral inactivation pool, may have a pH of about 7.40 to about 8.30, about 7.50 to about 7.70, about 7.55 to 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, It may be neutralized to a pH of 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.

[0367] In some embodiments, the sample may be further conditioned after neutralization and before contacting with the AEX material. The sample, e.g., a viral inactivation pool, may have a pH of about 3.00 mS / cm to about 6.00 mS / cm, about 3.00 mS / cm to about 4.00 mS / cm, about 3.40 mS / cm to 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 The sample may be adjusted to 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, about 5.50 mS / cm, about 5.60 mS / cm, about 5.70 mS / cm, about 5.80 mS / cm, about 5.90 mS / cm, or about 6.00 mS / cm. The sample may be adjusted, for example, using about 2 M sodium acetate, about 2 M Tris base, or a combination thereof.

[0368] In some embodiments, the AEX column is subjected to a pre-equilibration step before contact with the sample. The pre-equilibration step displaces strongly bound hydroxide ions on the AEX column to facilitate more rapid equilibration. In some exemplary embodiments, the pre-equilibration buffer (or "mobile phase") comprises about 2 M sodium chloride, WFI, or a combination thereof. In some exemplary embodiments, the linear velocity of the pre-equilibration buffer is about 100 to about 300 cm / hr, about 150 to 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 2 column volumes of pre-equilibration buffer are used.

[0369] In some embodiments, the AEX column is subjected to an equilibration step prior to contact with the sample, and optionally after a pre-equilibration step. The equilibration step changes the mobile phase to a pH and conductivity that matches the buffer excipients present in the load material (sample). In some embodiments, the equilibration buffer comprises about 50 mM Tris and about 60 mM acetic acid, at a pH of about 7.50 to about 7.70 and a conductivity of about 3.00 mS / cm to about 4.00 mS / cm. In some embodiments, the pH of the equilibration buffer is about 7.40 to about 8.30, about 7.50 to about 7.70, about 7.55 to 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. In some embodiments, the conductivity of the equilibration buffer is about 3.00 mS / cm to about 6.00 mS / cm, about 3.00 mS / cm to about 4.00 mS / cm, about 3.40 mS / cm to 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, S / 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, about 5.50 mS / cm, about 5.60 mS / cm, about 5.70 mS / cm, about 5.80 mS / cm, about 5.90 mS / cm, or about 6.00 mS / cm.In some exemplary embodiments, the linear velocity of the equilibration buffer is about 100 to about 300 cm / hr, about 150 to 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 3 column volumes of equilibration buffer are used.

[0370] In some embodiments, the concentration of protein loaded onto the AEX column ("AEX load," or grams 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, about 30.0 g / L, about 31.0 g / L, about 32.0 g / L, about 33.0 g / L, about 34.0 g / L, about 35.0 g / L, about 36.0 g / L, about 37.0 g / L, about 38.0 g / L, about 39.0 g / L, about 40.0 g / L, about 41.0 g / L, about 42.0 g / L, about 43.0 g / L, about 44.0 g / L, about 45.0 g / L, about 46.0 g / L, about 47.0 g / L, about 48.0 g / L, about 49.0 g / L, about 50.0 g / L, about 51.0 g / L, about 52.0 g / L, about 53.0 g / L, about 5 5.0 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 AEX loading is about 100 to about 300 cm / hr, about 150 to 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.

[0371] In some embodiments, the flow-through is collected when the UV absorbance at 280 nm reaches 0.2 AU on a 5 mm flow path, approximately 1.2 column volumes within the loading step, which is followed by a wash step to increase yield.

[0372] Wash steps are typically performed during AEX chromatography using conditions similar to the loading conditions, or by decreasing the pH and / or increasing the ionic strength / conductivity of the washes in a stepwise or linear gradient fashion. In one embodiment, the aqueous salt solutions used in both the loading and wash buffers have a pH at or near the isoelectric point (pI) of the protein of interest. Typically, the pH is about 0-2 units higher or lower than the pI of the protein of interest, but it can range from 0-0.5 units higher or lower. It can also be the pI of the protein of interest.

[0373] In some embodiments, the wash buffer comprises about 50 mM Tris and about 60 mM acetate, at a pH of about 7.50 to about 7.70, and a conductivity of about 3.00 mS / cm to about 4.00 mS / cm. In some embodiments, the pH of the wash buffer is about 7.40 to about 8.30, about 7.50 to about 7.70, about 7.55 to 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. In some embodiments, the conductivity of the wash buffer is from about 3.00 mS / cm to about 6.00 mS / cm, from about 3.00 mS / cm to about 4.00 mS / cm, from about 3.40 mS / cm to 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, or about 4.10 mS / cm. S / cm, approximately 4.20mS / cm, approximately 4.30mS / cm, approximately 4.40mS / cm, approximately 4.50mS / cm, approximately 4.60mS / cm, approximately 4.70mS / cm, approximately 4.80mS / cm, approximately 4.90mS / cm, approximately 5.00mS / cm, approximately 5.10 mS / cm, approximately 5.20mS / cm, approximately 5.30mS / cm, approximately 5.40mS / cm, approximately 5.50mS / cm, approximately 5.60mS / cm, approximately 5.70mS / cm, approximately 5.80mS / cm, approximately 5.90mS / cm, or approximately 6.00mS / cm.In some embodiments, the linear velocity of the wash buffer is about 100 to about 300 cm / hr, about 150 to 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 length of the wash is about 3 to about 5 column volumes (CV), about 3 CV, about 3.5 CV, about 4 CV, about 4.5 CV, or about 5 CV.

[0374] In some embodiments, the AEX column may be regenerated using one or more strip buffers. In some embodiments, a first strip buffer comprises 2 M sodium chloride (NaCl) and a second strip buffer comprises 1 N sodium hydroxide (NaOH). In some embodiments, the AEX column may be further soaked in 1 N NaOH for at least about 30 minutes.

[0375] In some embodiments, the AEX resin life is about 1 to about 100 cycles, about 50 to 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.

[0376] The anionic agent may be selected from the group consisting of acetate, chloride, formate, and combinations thereof. The cationic agent 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.

[0377] Packed anion exchange chromatography columns, anion exchange membrane devices, anion exchange monolith devices, or depth filter media can be operated in either bind-elute mode, flow-through mode, or hybrid mode in which proteins exhibit binding to the chromatographic material yet can be washed from such material using a buffer that is the same as or substantially similar to the loading buffer.

[0378] In the bind-and-elute mode, a column or membrane device is first conditioned with a buffer of appropriate ionic strength and pH under conditions that will cause a specific protein to adsorb to the resin-based matrix. For example, during feed loading, the protein of interest can be adsorbed to the resin by electrostatic attraction. After washing the column or membrane device with an equilibration buffer or another buffer with a different pH and / or conductivity, product recovery is achieved by increasing the ionic strength (e.g., conductivity) of the elution buffer to compete with the solute for the charged sites of the anion-exchange matrix. Changing the pH, thereby altering 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 (step elution).

[0379] In flow-through mode, a column or membrane device is operated at a selected pH and conductivity such that the protein of interest does not bind to the resin or membrane, while acidic species are retained on the column or have a distinct elution profile compared to the protein of interest. In the context of this strategy, acidic species, under appropriate conditions, will interact with or bind to the chromatographic material, while the protein of interest and certain aggregates and / or fragments of the protein of interest will flow through the column.

[0380] In some embodiments, the AEX step is performed in negative mode (flow-through mode), where negatively charged process-related impurities are adsorbed to the immobilized positively charged ligands and the protein of interest flows through.

[0381] Non-limiting examples of anion exchange resins include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Further non-limiting examples include: Poros 50PI and Poros 50H, which are rigid polymer beads with a backbone made 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 polymer-based beads; Fractogel® EMD TMAE Hicap, a synthetic polymer resin with tentacle ion exchangers; Sartobind STIC® PA nano, a salt-tolerant chromatography membrane with primary amine ligands; Sartobind Q nano, a strong anion-exchange chromatography membrane; CUNO BioCap, a Zeta Plus depth filter medium constructed with inorganic filter aid, purified cellulose, and ion exchange resin; XOHC, a depth filter medium constructed with inorganic filter aid, cellulose, and mixed cellulose esters; and Unosphere Q. In some embodiments, a resin is selected that has a relatively larger pore size to increase the surface area exposed to negatively charged species.

[0382] In some embodiments, the sample (batch) may be subsequently split into AEX steps, and the split batches may be further processed in parallel or serially. In some embodiments, the batch may not be split following the AEX step.

[0383] Additives such as polyethylene glycol (PEG), surfactants, amino acids, sugars, chaotropic agents, etc. may be added to enhance the performance of the separation and achieve better separation, recovery and / or product quality. Cation Exchange Chromatography

[0384] The method can include subjecting a biological sample containing the protein of interest to at least one cation exchange (CEX) step. In certain exemplary embodiments, the CEX step is in addition to the AEX step and can occur either before or after the AEX step. In some embodiments, the CEX is the third chromatographic unit operation in the purification process.

[0385] In carrying out cation exchange, a sample containing the protein of interest may be contacted with a cation exchange material using any of a variety of techniques, for example, by using batch production techniques or chromatography techniques, such as those described above for AEX. Cation exchange packed bed chromatography is based on ionic interactions between binding entities (target proteins or impurities) and functional groups immobilized on the chromatographic medium. Performance can be a function of, for example, the mobile phase, elution conditions, functional groups, and resin backbone. In some embodiments, a specific goal of the CEX step is to reduce HCP and HMW product-related impurities, such as protein aggregates.

[0386] In some embodiments, the concentration of protein loaded onto the CEX resin (e.g., grams protein per liter of resin) is about 40 g / L to 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.

[0387] In some embodiments, the CEX column is subjected to a pre-strip step to remove any bound impurities before starting a new separation. In some embodiments, the pre-strip buffer comprises about 2 M sodium chloride (NaCl). In some embodiments, about 2 column volumes of pre-strip buffer are used. In some embodiments, the pre-strip step is used only for the first cycle of a batch.

[0388] In some embodiments, the CEX column is subjected to an equilibration step to change the mobile phase pH and conductivity to support adsorption. In some embodiments, the equilibration buffer comprises about 40 mM sodium acetate at a pH of about 5.90 to about 6.10 and a conductivity of about 2.00 mS / cm to about 4.00 mS / cm. In some embodiments, the equilibration buffer comprises a pH of about 4.00 to about 6.50, about 5.00 to about 6.00, about 5.90 to 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 about 2.00 mS / cm to about 6.00 mS / cm, about 2.00 mS / cm to about 4.00 mS / cm, about 4.00 mS / cm to about 6.00 mS / cm, about 3.00 mS / cm to about 4.00 mS / cm, about 2.00 mS / cm, about 2.10 mS / cm, or about 2.20 mS / cm. S / cm, approx. 2.30mS / cm, approx. 2.40mS / cm, approx. 2.50mS / cm, approx. 2.60mS / cm, approx. 2.70mS / cm, approx. 2.80mS / cm, Approx. 2.90mS / cm, Approx. 3.00mS / cm, Approx. 3.10mS / cm, Approx. 3.20mS / cm, Approx. 3.30mS / cm, Approx. 3.40mS / cm, Approx. 3.50m S / cm, approx. 3.60mS / cm, approx. 3.70mS / cm, approx. 3.80mS / cm, approx. 3.90mS / cm, approx. 4.00mS / cm, approx. 4.10mS / cm, Approx. 4.20mS / cm, Approx. 4.30mS / cm, Approx. 4.40mS / cm, Approx. 4.50mS / cm, Approx. 4.60mS / cm, Approx. 4.70mS / cm, Approx. 4.80m S / 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, about 5.50 mS / cm, about 5.60 mS / cm, about 5.70 mS / cm, about 5.80 mS / cm, about 5.90 mS / cm, or about 6.00 mS / cm. In some embodiments, about 3 column volumes of equilibration buffer are used.

[0389] The sample (CEX load) may be conditioned prior to contact with the CEX material. In some embodiments, the sample, e.g., the AEX pool, is adjusted to a pH of about 4.00 to about 6.50, about 5.00 to about 6.00, about 5.90 to 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 2M acetic acid.

[0390] In some embodiments, the CEX column is subjected to a wash step to remove weakly bound impurities. In some embodiments, the equilibration buffer is also used as the wash buffer. In some embodiments, the wash buffer comprises about 40 mM sodium acetate at a pH of about 5.90 to about 6.10 and a conductivity of about 2.00 mS / cm to about 4.00 mS / cm. In some embodiments, the wash buffer comprises a pH of about 4.00 to about 6.50, about 5.00 to about 6.00, about 5.90 to 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 from about 2.00 mS / cm to about 6.00 mS / cm, from about 2.00 mS / cm to about 4.00 mS / cm, from about 4.00 mS / cm to about 6.00 mS / cm, from about 3.00 mS / cm to about 4.00 mS / cm, about 2.00 mS / cm, about 2.10 mS / cm, or about 2.20 mS / cm. / cm, approx. 2.30mS / cm, approx. 2.40mS / cm, approx. 2.50mS / cm, approx. 2.60mS / cm, approx. 2.70mS / cm, approx. 2.80mS / cm, approx. 2.90mS / cm, approx. 3.00mS / cm, approx. 3.10mS / cm, approx. 3.20mS / cm, approx. 3.30mS / cm, approx. 3.40mS / cm, approx. 3.50mS / cm, approx. 3.60mS / cm, approx. 3.70mS / cm, approx. 3.80mS / cm, approx. 3.90mS / cm, approx. 4.00mS / cm, approx. 4.10mS / cm, approx. 4.20mS / cm, approx. 4.30mS / cm, approx. 4.40mS / cm, approx. 4.50mS / cm, approx. 4.60mS / cm, approx. 4.70mS / cm, approx. 4.80m S / 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, about 5.50 mS / cm, about 5.60 mS / cm, about 5.70 mS / cm, about 5.80 mS / cm, about 5.90 mS / cm, or about 6.00 mS / cm. In some embodiments, about 2 column volumes of wash buffer are used.In some embodiments, the wash buffer comprises potassium sorbate.

[0391] After the wash step, the protein of interest may be eluted by increasing the conductivity. In some embodiments, CEX pool collection begins at 0.2 A using a 2 mm UV path and ends after approximately 5 column volumes (CV) of pool collection. In some embodiments, the elution buffer comprises about 20 mM Tris and about 120 mM sodium acetate at a pH of about 5.90 to about 6.20 and a conductivity of about 9.00 mS / cm to about 11.00 mS / cm. In some embodiments, the elution buffer comprises a pH of about 5.70 to about 7.00, about 5.90 to 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 about 7.00 mS / cm to about 11.00 mS / cm, about 9.00 mS / cm to about 11.00 mS / cm, about 10.00 mS / cm to 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 7.50 mS / cm, about 7.60 mS / cm, about 7.70 mS / cm, about 7.80 mS / cm, about 7.90 mS / cm, about 8.00 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, or about 8.60 mS / cm , approx. 8.70mS / cm, approx. 8.80mS / cm, approx. 8.90mS / cm, approx. 9.00mS / cm, approx. 9.10mS / cm, approx. 9.20mS / cm, approx. 9. 30mS / cm, approx. 9.40mS / cm, approx. 9.50mS / cm, approx. 9.60mS / cm, approx. 9.70mS / cm, approx. 9.80mS / cm, approx. 9.90mS / cm, about 10.00 mS / cm, about 10.10 mS / cm, about 10.20 mS / cm, about 10.30 mS / cm, about 10.40 mS / cm, about 10.50 mS / cm, about 10.60 mS / cm, about 10.70 mS / cm, about 10.80 mS / cm, about 10.90 mS / cm, or about 11.00 mS / cm. In some embodiments, about 5 column volumes of elution buffer are used.

[0392] In some embodiments, after elution, the CEX column is subjected to a high ionic strength strip followed by a caustic strip to remove bound impurities and prepare the resin for further cycles or storage. In some embodiments, the first strip buffer comprises about 2 M sodium chloride (NaCl) and the second strip buffer comprises about 1 N sodium hydroxide (NaOH).

[0393] In some embodiments, each separation phase, excluding elution, is performed at a linear velocity of about 100 to about 300 cm / hr, about 150 to 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 separation phase, excluding elution, is performed at a linear velocity of 200 cm / hr. In an exemplary embodiment, the elution step is carried out at a linear velocity of 100 cm / hr.

[0394] In some embodiments, the CEX resin life is about 1 to about 100 cycles, about 50 to 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.

[0395] An aqueous salt solution may be used as both the loading and washing buffer, having a pH lower than the isoelectric point (pI) of the protein of interest. In one embodiment, the pH is about 0-5 units lower than the protein's pI. In another embodiment, it is in the range of about 1-2 units lower than the protein's pI. In yet another embodiment, it is in the range of about 1-1.5 units lower than the protein's pI.

[0396] In certain exemplary embodiments, the concentration of the anionic agent in the aqueous salt solution is increased or decreased to achieve a pH of about 3.5 to about 10.5, or about 4 to about 10, or about 4.5 to about 9.5, or about 5 to about 9, or about 5.5 to about 8.5, or about 6 to about 8, or about 6.5 to about 7.5. In one aspect, the concentration of the anionic agent is increased or decreased in the aqueous salt solution to achieve a pH of 5, or 5.5, or 6, or 6.5, or 6.8, or 7.5. Buffer systems suitable for use in the CEX method include, but are not limited to, Tris formate, Tris acetate, ammonium sulfate, sodium chloride, and sodium sulfate.

[0397] In certain exemplary embodiments, the conductivity and pH of the aqueous salt solution are adjusted by increasing or decreasing the concentration of a cationic agent. In one aspect, the cationic agent is maintained at a concentration ranging from about 20 mM to about 500 mM, from about 50 mM to about 350 mM, from about 100 mM to about 300 mM, or from about 100 mM to about 200 mM. Non-limiting examples of cationic agents may be selected from the group consisting of sodium, Tris, triethylamine, ammonium, arginine, and combinations thereof.

[0398] Packed cation exchange chromatography columns or cation exchange membrane devices can be operated in either bind-and-elute mode, flow-through mode, or hybrid mode, in which the product binds to or interacts with the chromatographic material and can 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). In some embodiments, the CEX step is performed in positive (bind-and-elute) mode, in which the positively charged protein of interest and impurities are adsorbed to an immobilized, negatively charged stationary phase. The protein of interest is then eluted, while many impurities remain bound to the stationary phase. A series of regeneration steps removes the bound impurities and prepares the CEX column for the next cycle.

[0399] Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S) groups. Additional cationic materials include, but are not limited to: Capto SP ImpRes, which are high-flow agarose beads; Capto S ImpAct; CM Hyper D grade F, which are functionalized hydrogel, permeabilized ceramic beads coated with 250-400 μeq / mL of ionic groups; Eshmuno S, which is a hydrophilic polyvinyl ether-based matrix with 50-100 μeq / mL ionic capacity; Nuvia C Prime, which is a hydrophobic cation exchange medium composed of a macroporous, highly cross-linked hydrophilic polymer matrix with 55-75 με / mL; Nuvia S, which has a UNOsphere-based matrix with 90-150 με / mL of ionic groups; Poros HS, which are rigid polymer beads with a backbone composed of cross-linked poly[styrene-divinyl-benzene]; Poros XS, which are rigid polymer beads with a backbone composed of cross-linked poly[styrene-divinyl-benzene]; Toyo Pearl Giga Cap CM 650, which are polymer-based beads with 0.225 meq / mL ionic capacity; and Toyo Pearl Giga Cap S, which are polymer-based beads. 650M; Toyo Pearl MX TRP, a polymer-based bead; and Fractogel® EMD SE Hicap. It is noted that CEX chromatography can be used with the MM resins described herein. In some embodiments, the CEX resin is selected to allow for a relatively higher capacity, e.g., up to a maximum loading of about 100 g / L of resin. In some embodiments, the CEX resin is selected for relatively higher caustic stability, e.g., to prevent hydrolysis of cationic materials.

[0400] Additives such as polyethylene glycol, surfactants, amino acids, sugars, and chaotropic agents may be added to enhance the performance of the separation so that better separation, recovery, and / or product quality is achieved. Mixed-Mode Chromatography

[0401] Mixed-mode ("MM") chromatography can also be used in the process of the present invention. MM chromatography, also referred to herein as "multimode chromatography" or "MMC," is a chromatographic strategy that utilizes a support containing ligands 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-charge interaction between the ligand and the protein of interest, while the other side provides an electron acceptor-donor interaction and / or a hydrophobic and / or hydrophilic interaction. Electron donor-acceptor interactions include hydrogen bonding, π-π, cation-π, charge transfer, dipole-dipole, induced dipole, etc.

[0402] The column resin used for mixed-mode separation may be Capto Adhere. Capto Adhere is a strong anion exchanger with multimodal functionality. Its base matrix is ​​highly cross-linked agarose containing a ligand (N-benzyl-N-methylethanolamine) that exhibits different functionalities due to interactions, such as ionic interactions, hydrogen bonding, and hydrophobic interactions. In certain embodiments, the resin used for mixed-mode separation is selected from PPA-HyperCel and HEA-HyperCel. The base matrix of PPA-HyperCel and HEA-HyperCel is porous cross-linked cellulose. The ligands are phenylpropylamine and hexylamine, respectively. Phenylpropylamine and hexylamine offer different selectivity and hydrophobicity options for protein separation. Additional mixed-mode chromatography supports include Capto MMC, MEP-HyperCel, MBI HyperCel, CMM HyperCel, Capto Adhere ImpRes, Capto Core 700, Nuvia C Prime, Toyo Pearl MX Trp 650M, and Eshmuno® HCX. In certain embodiments, mixed-mode chromatography resins are composed of ligands coupled directly or through spacers to an organic or inorganic support, sometimes represented by a base matrix. The support may be in the form of particles, e.g., essentially spherical particles, monoliths, filters, membranes, surfaces, capillaries, etc. In certain embodiments, the support is prepared from natural polymers, e.g., cross-linked carbohydrate materials, such as agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginic acid, etc. To achieve high adsorption capacity, the support may be porous, and then ligands are coupled to the exterior surface as well as the pore surfaces. Such natural polymer supports can be prepared according to standard methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964), the entire disclosure of which is incorporated herein by reference).Alternatively, the support may be prepared from a synthetic polymer, for example, a cross-linked synthetic polymer, such as styrene or a styrene derivative, divinylbenzene, acrylamide, acrylic acid esters, methacrylic acid esters, vinyl esters, vinylamides, etc. Such synthetic polymers may be produced according to standard methods. See R. Arshady, "Styrene-based polymer supports developed by suspension polymerization" (R. Arshady, Chimica e L'Industria 70(9), 70-75 (1988 (1964)), the entire disclosure of which is incorporated herein by reference). Porous natural or synthetic polymer supports are also available from commercial sources, such as Cytiva, Uppsala, Sweden. Depending on the resin used, the MMC may be operated in flow-through mode or in bind-and-elute mode; for example, AEX / HIC hybrid resins are preferably operated in flow-through mode, while CEX / HIC hybrid resins may be preferably operated in bind-and-elute mode.

[0403] Additives such as polyethylene glycol, surfactants, amino acids, sugars, chaotropic agents may be added to enhance separation performance to achieve better separation, recovery and / or product quality.

[0404] The disclosed method may also be performed in a sequential chromatography mode, in which at least two columns are used (referred to as the "first" column and the "second" column). In certain embodiments, the sequential chromatography mode may be performed such that the eluted and / or stripped fractions can then be loaded onto the second column sequentially or simultaneously (with or without dilution).

[0405] In one embodiment, the media choice for continuous mode can be one of a variety of chromatographic resins with pendant hydrophobic and anion exchange functional groups, monolith media, membrane adsorption media, or depth filtration media.

[0406] In some exemplary embodiments, MMC is the second chromatographic separation in the process, following affinity capture chromatography, optionally followed by an AEX step. In other embodiments, MMC is the third chromatographic separation in the process, following affinity capture chromatography and an AEX step. In some embodiments, the methods of the present disclosure include MMC and may not include a CEX or HIC step. In some embodiments, a process for producing a recombinant protein such as dupilumab includes two, three, or four chromatographic modes, including affinity capture chromatography, MMC, and optionally an AEX, CEX, HIC, or additional MMC step, in any order before or after MMC. MMC can be operated in flow-through mode or bind-and-elute mode, regardless of the order of the chromatographic steps. MMC can be operated in a plate-based format, such as a 96-well plate-based format, or a robocolumn-based format.

[0407] In some embodiments, the MMC column is subjected to an equilibration step to change the mobile phase pH and conductivity to support adsorption. In some embodiments, the equilibration buffer contains about 100 mM NaCl at a pH of about 5. In some embodiments, the equilibration buffer contains about 4.50 to about 9.00, about 4.50 to about 8.00, about 4.50 to about 5.50, about 5.00 to 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, or 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.

[0408] In some embodiments, the equilibration buffer may be about 100 mM to about 500 mM, about 100 mM to about 250 mM, about 100 mM to about 150 mM, about 80 mM to about 120 mM, about 95 mM to about 105 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, The equilibration buffer may contain about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, about 475 mM, or about 500 mM NaCl. In some embodiments, the equilibration buffer contains about 0 mM to about 100 mM NaCl. In some embodiments, the equilibration buffer contains citric acid or arginine.

[0409] In some embodiments, the wash buffer can include any of the pH values, NaCl, concentrations, or buffer salts of the equilibration buffers described above.

[0410] In some embodiments, the amount of protein loaded onto the MMC resin (e.g., grams protein per liter of resin) is about 100 g / L or about 110 g / L. In some embodiments, the amount of protein loaded onto the MMC resin is about 50 g / L to about 200 g / L, about 100 g / L to about 150 g / L, about 100 g / L to 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, or about 100 g / L. L, approximately 105g / L, approximately 110g / L, approximately 115g / L, approximately 120g / L, approximately 125g / L, approximately 130g / L, approximately 135g / L, approximately 140g / L, approximately 145g / L, approximately 150g / L, approximately 1 55g / L, about 160g / L, about 165g / L, about 170g / L, about 175g / L, about 180g / L, about 185g / L, about 190g / L, about 195g / L, or about 200g / L. In some embodiments, the amount of protein loaded onto the MMC resin is about 10 g / L to 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.

[0411] In some embodiments, the MMC is operated in a bind and elute mode, which further includes the use of an elution buffer. In some embodiments, the elution buffer has a pH of about 4.50 to about 9.00, about 4.50 to about 8.00, about 4.50 to about 5.50, about 5.00 to 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, or Including a pH of 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.

[0412] In some embodiments, the elution buffer is from about 0 mM to about 500 mM, from about 100 mM to about 250 mM, from about 100 mM to 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, about 10 mM In some embodiments, the elution buffer comprises 0 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, about 475 mM, or about 500 mM NaCl. In some embodiments, the elution buffer comprises citrate or arginine. Hydrophobic Interaction Chromatography

[0413] The disclosed methods can include subjecting a biological sample containing a protein of interest to at least one hydrophobic interaction chromatography (HIC) step. In performing the separation, the biological sample is contacted with an HIC material, for example, using batch production techniques or using column or membrane chromatography. Prior to HIC processing, it may be desirable to adjust the concentration of the salt buffer to achieve the desired protein binding / interaction to 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.

[0414] In some embodiments, a particular objective of the HIC step may be to reduce the level of HCP or HMW product-related impurities, including, for example, PLBD2. PLBD2 levels in the HIC load may be present at 100-300 ppm and may be reduced to 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, to less than 100 ppm, less than 30 ppm, less than 4 ppm, or less than 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.

[0415] In some embodiments, a batch of sample may be split into two sublots, which may be processed individually through an HIC step and subsequent steps, such as virus-retentive filtration and UF / DF, before being recombined, e.g., prior to a third-stage concentration.

[0416] 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. Hydrophobic groups on or within the protein interact with hydrophobic groups on the chromatography resin or membrane. Typically, under appropriate conditions, the more hydrophobic the protein, the stronger its interaction with the column or membrane. Therefore, under appropriate 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.

[0417] Similar to ion exchange chromatography, HIC columns or HIC membrane devices may also be operated in elution mode, flow-through, or hybrid mode, in which the product binds to or interacts with the chromatographic material but can still 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 in connection with AEX processing). In some embodiments, the HIC step is performed in negative mode, in which process-related impurities bind to the immobilized ligand and the protein of interest flows through.

[0418] Because hydrophobic interactions are strongest at high ionic strength, this type of separation is preferably performed after a salt elution step, such as those typically used in conjunction with ion-exchange chromatography. Alternatively, salt may be added to the sample before the HIC step is used. Protein adsorption to the HIC column is favored by high salt concentrations, but the actual concentration can vary over a wide range depending on the nature of the protein of interest, the salt type, and the particular HIC ligand selected. Various ions may be placed in a so-called soluphobic series, depending on whether they enhance hydrophobic interactions (salting-out effect) or weaken them by disrupting the structure of water (chaotropic effect). Cations are associated with an increased salting-out effect, while Ba2+ ;Ca 2+ ;Mg 2+ ;Li + ;Cs + ;Na + ;K + ;Rb + ;NH4 + On the other hand, anions are associated with an increased chaotropic effect, and PO4 3- ;SO4 2- ;CH3CO3 - ;CI - ;Br - ;NO3 - ;ClO4 - ;I - ;SCN - It can be ranked as.

[0419] HIC media typically contain a base matrix (e.g., cross-linked agarose or synthetic copolymer materials) to which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled. Suitable HIC media include agarose resins or membranes functionalized with phenyl groups (e.g., Cytiva's Phenyl Sepharose™ or Sartorius' Phenyl Membrane). A variety of HIC resins are commercially available. Examples include, but are not limited to, Capto Phenyl, Capto Butyl, low- or high-substituted Phenyl Sepharose™ 6 Fast Flow, Phenyl Sepharose™ High Performance, Octyl Sepharose™ High Performance (GE Healthcare); Fractogel™ EMD Propyl or Fractogel™ EMD Phenyl (E. Merck, Germany); Macro-Prep™ Methyl or Macro-Prep™ t-Butyl columns (Bio-Rad, California); WP HI-Propyl (C3)™ (JT Baker, New Jersey); and Toyopearl™ ether, phenyl, or butyl (TosoHaas, PA); Toyo PPG; Toyo Phenyl; Toyo Butyl; and Toyo Hexyl.

[0420] The pH selected for any particular production process must be compatible with protein stability and activity, so the specific pH conditions will be specific to each application. However, between pH 5.0 and 8.5, certain pH values ​​may be preferred because they have very little impact on the final selectivity and resolution of the HIC separation. Increasing pH weakens hydrophobic interactions, and protein retention changes more dramatically at pH values ​​above 8.5 or below 5.0. Furthermore, changes in ionic strength, the presence of organic solvents, temperature, and pH (especially at the isoelectric point, pI, where there is no net surface charge) can affect protein structure and solubility and, consequently, interactions with other hydrophobic surfaces, such as those in the HIC medium. Thus, in certain embodiments, the present disclosure incorporates production strategies in which one or more of the foregoing are adjusted to achieve the desired reduction in process-related impurities and / or product-related substances.

[0421] In certain exemplary embodiments, spectroscopic methods such as UV, NIR, FTIR, fluorescence, and Raman may be used to monitor proteins of interest and impurities in online, at-line, or in-line modes, which may then be used to control the level of aggregates in the pooled material collected from the HIC adsorption effluent. In other exemplary embodiments, online, at-line, or in-line monitoring methods may be used either on the effluent line of the chromatography step or in the collection vessel to enable the achievement of desired product quality / recovery. In still other exemplary embodiments, the UV signal may be used as a surrogate to achieve appropriate product quality / recovery, and processing techniques such as integration, differential, and moving average may be included to appropriately process the UV signal to address normal process variability and ensure that target product quality is achieved. In certain exemplary embodiments, such measurements may be combined with in-line dilution methods to control load / wash ion concentrations / conductivities by feedback, thus facilitating product quality control.

[0422] 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 120 g / L. g / L, approximately 105g / L, approximately 110g / L, approximately 115g / L, approximately 120g / L, approximately 125g / L, approximately 130g / L, approximately 135g / L, approximately 140g / L, approximately 145g / L, approximately 150g / L, approximately 155g / L, about 160g / L, about 165g / L, about 170g / L, about 175g / L, about 175g / L, about 180g / L, about 185g / L, about 190g / L, about 195g / L, or about 200g / L.

[0423] In some embodiments, the sample may be conditioned prior to contact with the HIC material, which may be referred to as a load conditioning. In some embodiments, the sample, e.g., the CEX pool, is adjusted to about 40 mM citric acid using a single bolus addition of about 1.2 M sodium citrate. After conditioning, the pH of the sample (HIC load) is about 6.30 to about 6.70, and the conductivity of the sample is about 14.00 to about 17.00 mS / cm. In some embodiments, the HIC load comprises a pH of about 5.50 to about 7.00, about 6.30 to 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 is from about 14.00 mS / cm to 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, and a conductivity of 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 during HIC loading is about 10 mM to about 50 mM, about 30 mM to 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.

[0424] In some embodiments, the HIC column is subjected to a pre-equilibration step before contacting it with the sample. The pre-equilibration step removes any 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 about 100 to about 300 cm / hr, about 150 to 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 exemplary embodiments, the linear velocity is about 200 cm / hr. In some exemplary embodiments, about 3 column volumes of pre-equilibration buffer are used.

[0425] In some embodiments, the HIC column is subjected to an equilibration step before contacting with the sample, and optionally after a pre-equilibration step. The equilibration step changes the mobile phase to a pH and conductivity that matches the buffer excipients present in the load material (sample). In some embodiments, the equilibration buffer can include about 40 mM Tris and about 40 mM sodium citrate, at a pH of about 6.30 to about 6.70, and a conductivity of about 8.50 mS / cm to about 12.00 mS / cm. In some embodiments, the linear velocity of the equilibration buffer is about 100 to about 300 cm / hr, about 150 to 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 exemplary embodiments, the linear velocity is about 200 cm / hr. In some exemplary embodiments, about 2 column volumes of equilibration buffer are used.

[0426] In some embodiments, the protein concentration of the HIC load is about 10.0 g / L to about 20.0 g / L, about 12.0 g / L to 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 velocity of HIC loading is about 100 to about 300 cm / hr, about 150 to 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.

[0427] In some embodiments, the flow-through is collected when the UV absorbance at 280 nm reaches 0.2 AU on a 5 mm flow path, approximately 3-4 column volumes within the loading step, followed by a wash step to increase yield.

[0428] In some embodiments, the equilibration buffer is also used as a wash buffer, ie, the wash buffer comprises about 40 mM Tris and about 40 mM sodium citrate, at a pH of about 6.30 to about 6.70, and a conductivity of about 8.50 mS / cm to about 12.00 mS / cm. In some embodiments, the linear velocity of the wash buffer is about 100 to about 300 cm / hr, about 150 to 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 about 6 to about 8 column volumes (CV), about 6 CV, about 6.5 CV, about 7 CV, about 7.5 CV, or about 8 CV. In an exemplary embodiment, the wash length is about 8 CV.

[0429] In some embodiments, the HIC column can be regenerated using one or more strip buffers. In some embodiments, a first strip buffer contains about 2 column volumes of WFI, a second strip buffer contains about 2 column volumes of 1 N sodium hydroxide, and a third strip buffer contains about 2 column volumes of WFI. The strip buffer contains 5 mM sodium hydroxide. Additional steps may be performed for the final cycle of the batch. In some embodiments, the final cycle of the batch includes an additional strip step using about 2 column volumes of about 20% ethanol, an additional strip step using about 2 column volumes of WFI, and a final equilibration step performed as described above.

[0430] In some embodiments, the HIC resin life is about 1 to about 100 cycles, about 50 to 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. Size Exclusion Chromatography

[0431] Size exclusion chromatography, or gel filtration, relies on the separation of components as a function of molecular size. Separation depends on the amount of time a substance spends in a porous stationary phase compared to its time in a fluid. The likelihood of a molecule residing in a pore depends on the size of the molecule and the pore. Furthermore, the ability of a substance to penetrate the interior of a pore is determined by the diffusional mobility of the macromolecule, which is higher for small macromolecules. Very large macromolecules may not be able to penetrate the pores of the stationary phase at all; for very small macromolecules, the likelihood of penetration is fairly consistent. Larger molecular-sized components migrate more rapidly through the stationary phase, while smaller molecular-sized components migrate longer paths through the pores of the stationary phase and are therefore retained longer in the stationary phase.

[0432] The chromatography material may include a resin or membrane size-exclusion material. 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 swollen gel beads. Molecules larger than a certain size will not enter the gel beads and will therefore move fastest through the chromatographic bed. Smaller molecules, such as surfactants, proteins, DNA, etc., will enter the gel beads to varying degrees depending on their size and shape, and their passage through the bed will be retarded. Thus, molecules are generally eluted in order of decreasing molecular size.

[0433] Porous chromatography resins suitable for size-exclusion chromatography of viruses can be made of dextrose, agarose, polyacrylamide, or silica, which have different physical properties. Combinations of polymers can also be used. The most commonly used is available from Amersham Biosciences under the trade name "SEPHADEX." Other size-exclusion supports of different construction materials are also suitable, such as Toyopearl 55F (polymethacrylate, Tosoh Bioscience, Montgomery Pa.) and Bio-Gel P-30 Fine (BioRad Laboratories, Hercules, CA). Virus-retaining filtration

[0434] Viral filtration is a specialized virus reduction step during the production process. This step is usually performed after chromatographic polishing. Viral reduction by virus-retentive membranes is based on the difference in hydrodynamic radius, where the protein of interest, e.g., a monoclonal antibody (hydrodynamic radius of approximately 10 nm), passes through the filter, while larger viruses (hydrodynamic radius greater than 18 nm) are retained by the membrane.

[0435] Viral reduction can be achieved through the use of appropriate filters, including, but not limited to, Asahi Kasei Pharma's Planova 20N™, 50N, or BioEx, EMD Millipore's Viresolve™ filters, Sartorius' ViroSart® CPV or Virosart® HF, or Pall Corporation's Ultipor DV20, DV50™, or Pegasus Prime filters. It will be apparent to one of ordinary skill in the art to select an appropriate filter to obtain the desired filtration performance.

[0436] The virus-retentive filtration step may further include a prefilter step. Virus-retentive filters are vulnerable to premature fouling due to pore blockage caused by impurities present in the feed stream. Fouling is undesirable because the associated virus-retentive filter flux attenuation has been linked to viral breakthrough for certain virus-retentive filters. Studies have reported that the use of guards or prefilters can mitigate virus filter fouling. Chemically functionalized prefilters, including HIC, CEX, and AEX types, have been found by the inventors to improve virus-retentive filter capacity. Prefiltering can be achieved by using an appropriate prefilter, including, but not limited to, Viresolve Shield, Viresolve Shield H, Millistak+ HC Pro X0SP (MilliporeSigma), or Pall Corporation's Pegasus Protect. Those of ordinary skill in the art will readily appreciate the ability to select an appropriate prefilter to achieve the desired filtration performance.

[0437] In some embodiments, a virus-retaining filtration (VRF) step is performed immediately after the HIC step. In some embodiments, the load material is conditioned with three pre-filters immediately preceding the three filters.

[0438] In some exemplary embodiments, the VRF process comprises 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 is to prime and wet the device with 50 L / m at an operating TMP of 25±5 psi. 2 This involves a flush greater than WFI. This is followed by a pre-use integrity test using sterile oil-free air or nitrogen gas. The fourth step involves a buffer flush and flow verification, where the assembly is flushed with 20 L / m at 25±5 psi TMP. 2Equilibrate with a larger volume of a buffer containing about 40 mM Tris and about 40 mM sodium citrate, pH 6.5.

[0439] The sample, e.g., the HIC pool, is then pumped through a constant 25±5 psi TMP, 900 L / m 2 The sample is loaded at a volume of less than 700 L / m. Pool collection begins immediately after loading begins and ends when the load material is depleted. In some embodiments, the sample volume is about 700 L / m. 2 ~Approx. 1300L / m 2 , 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 approximately 2000 L / m 2In some embodiments, the load conductivity is about 7.5 mS / cm to 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 load pH is about 4.5 to about 7.5, about 6.3 to 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 about 4.5 g / L to about 13 g / L, about 8 g / L to 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.

[0440] Finally, the filters are flushed with WFI in the preparation using sterile oil-free air or nitrogen gas for a final post-use integrity test. Concentration and Diafiltration

[0441] Certain exemplary embodiments of the present disclosure use ultrafiltration and diafiltration (UF / DF), also referred to as concentration and diafiltration, to further concentrate and formulate the protein of interest. UF / DF conditions the drug substance to achieve a pH, excipient content, and protein concentration conducive to long-term storage and the addition of stabilizing excipients to produce the final drug substance (FDS). During processing, the protein solution is pumped tangentially across the surface of a semipermeable plane-parallel sheet membrane. The membrane may have a pore size of approximately 50 kDa, for example, permeable to water and buffer salts but generally impermeable to the protein of interest, such as a monoclonal antibody. At the exit of the membrane flow channel, a driving force for osmosis is applied at a transmembrane pressure (TMP) induced by the flow restriction.

[0442] 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 Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9), the entire contents of which are incorporated herein by reference. One filtration process is tangential flow filtration, as described in the Millipore catalog entitled "Pharmaceutical Process Filtration Catalogue", pp. 177-202 (Bedford, Mass., 1995 / 96), the entire contents of which are incorporated herein by reference. Ultrafiltration is generally considered to mean filtration using a filter with a pore size of less than 0.1 μm. By using filters with such small pore sizes, it is possible to reduce the sample volume by sample buffer penetration through the filter membrane pores, while proteins are retained on the membrane surface.

[0443] Typically, there are three to four UF / DF stages: first concentration, diafiltration, secondary concentration, and final concentration. During the first concentration, the TMP drives water and salts across a permeable membrane, which reduces the liquid volume and therefore increases the protein concentration. The concentration level of the first concentration can be optimized to balance throughput and protein stability.

[0444] During the diafiltration stage, permeable solutes are displaced as new buffer is washed into the product stream. When new buffer is added at the same rate as permeate is removed from the system, the sum of the retentate tank and skid volumes defines the system volume. One turnover volume (TOV) is defined as the amount of diafiltration buffer added to the UF / DF process and matches the system volume. Typically, eight system volume displacements (8 TOV) ensure >99.9% buffer exchange. The diafiltration buffer is intended to condition the protein to a stable pH and excipient concentration that ensures high product concentration.

[0445] Once adequate buffer exchange has been achieved, secondary concentration further reduces the product volume to facilitate storage and formulation. Final concentration is performed if further product volume reduction is required, for example, due to plant compatibility considerations, higher final protein concentration requirements, or adjustment needs with viscosity control agents.

[0446] Those of ordinary skill in the art can select an appropriate membrane filter device for UF / DF operation. Examples of membrane cassettes suitable for the present invention include, but are not limited to, EMD Millipore's Pellicon 2 or Pellicon 3 cassettes with 10 kD, 30 kD, or 50 kD membranes, Cytiva's Kvick 10 kD, 30 kD, or 50 kD membrane cassettes, and Pall Corporation's Centramate or Centrasette 10 kD, 30 kD, or 50 kD cassettes.

[0447] Exemplary steps are described in more detail herein. The final UF / DF step is performed at a flow rate of approximately 12 L / m for the retentate. 2 , and 40 L / m for permeate 2 The feed flow rate is approximately 200-300 L / hr / m at a pressure of approximately 25 ± 5 psi. 2 (LMH). A pre-use leak test is then performed with WFI at an inlet pressure of 8-12 psi. Equilibration is then performed with approximately 4 mM acetic acid at approximately pH 4.10±0.10. The volume of the equilibration buffer is approximately 5 L / m 2 The flow rate is about 200 to 300 LMH.

[0448] The sample to be concentrated (load) is then adjusted for compatibility with the final drug substance composition. The load adjustment solution can be, for example, about 10% (w / v) ultra-purified polysorbate 80 or polysorbate 20 added at about 50 μL / L load.

[0449] A first concentration step is then performed on the load, e.g., VRF pool. The load is approximately 970 g / m 2 The following volume is added, 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 the initial concentration can be about 60 to 80 g / L. In an exemplary embodiment, the protein concentration at this step is about 70 g / L.

[0450] The initial concentration step is followed by a diafiltration step. The diafiltration buffer can contain about 4 mM acetic acid at a pH of about 4.00 to 4.20. In some embodiments, the acetic acid concentration in the diafiltration buffer is about 3 mM to about 10 mM, about 3 mM to 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 about 4.00 to 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.

[0451] The volume of diafiltration buffer used can be 8 TOV or more, with a flow rate of about 200-350 LMH and a pressure of about 10-30 psi. In an exemplary embodiment, the diafiltration flow rate is about 300 LMH. In an exemplary embodiment, the diafiltration pressure is about 20 psi. The protein concentration after diafiltration can be about 60 to about 80 g / L. In an exemplary embodiment, the protein concentration after diafiltration is about 70 g / L.

[0452] Diafiltration is followed by a secondary concentration step. The product mass used for secondary concentration is approximately 970 g / m 2 The flow rate of the secondary concentration is about 100 to about 350 LMH. In an exemplary embodiment, the flow rate is about 300 LMH. The pressure of the secondary concentration is about 10 to 30 psi, with an inlet pressure of less than 60 psi. The protein concentration after the secondary concentration can be about 80 to 100 g / L. In an exemplary embodiment, the protein concentration after the secondary concentration is about 90 g / L.

[0453] After the secondary concentration is the membrane depolarization step. This process can have a duration of approximately 5-15 minutes, with a flow rate of 350 LMH or less and a pressure of less than 30 psi. For this step, the permeate valve is 0% open, while the retentate valve is 100% open.

[0454] Next comes the final or third concentration step. The amount of final concentrate charge used is about 1800 g / m, added at a flow rate of about 5-350 LMH and an inlet pressure of less than 60 psi. 2 or less. In some embodiments, the final protein concentration is about 180 g / L to about 260 g / L, about 225 g / L to about 255 g / L, about 228 g / L to about 232 g / L, about 180 g / L, about 185 g / L, about 190 g / L, about 195 g / L, about 200 g / L, about 205 g / L, about 210 g / L, about 215 g / L, about 220 g / L, about 225 g / L, about 230 g / L, about 235 g / L, about 240 g / L, about 245 g / L, about 250 g / L, about 255 g / L, or about 260 g / L. In an exemplary embodiment, the final protein concentration is about 232 g / L. In another exemplary embodiment, the final protein concentration is about 240 g / L.

[0455] After final concentration, the process includes another membrane depolarization step, which occurs for approximately 15-35 minutes. In an exemplary embodiment, this step has a duration of approximately 25 minutes. The feed flow rate is approximately 350 LMH or less, the pressure is less than 30 psi, the permeate valve is 0% open, and the retentate valve is 100% open.

[0456] Finally, after membrane polarization, there was a WFI rinse step, with 10 L / m 2 WFI is applied at a flow rate of about 200 to about 250 LMH. combination

[0457] Certain exemplary embodiments include further conditioning the final concentrate pool (FCP) protein after the concentration and diafiltration steps, adjusting the concentrated protein to the desired concentration and with the desired excipients for the final drug substance (FDS).

[0458] If the filtered FCP protein concentration is higher than the specified target, adjust to the target concentration by diluting with FCP dilution buffer, which may contain 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 FCP dilution buffer can be about 2.0 mM to about 14.0 mM, about 6.0 mM to about 12.0 mM, about 8.0 mM to 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 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11.0 mM, about 11.5 mM, about 12.0 mM, about 12.5 mM, about 13.0 mM, about 13.5 mM, or about 14.0 mM. In some embodiments, the concentration of arginine hydrochloride in the FCP dilution buffer can be about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to 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 about 1% to 10%, about 2% to 9%, about 3% to 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 about 4.0 to about 6.5, about 4.5 to about 6.0, about 5.0 to 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.

[0459] In some embodiments, the FCP may be further combined with an excipient concentrated buffer to achieve a target dilution of the FCP protein and to achieve a target excipient concentration. The FCP excipient concentrated buffer may comprise about 10 mM acetic acid, about 350 mM arginine hydrochloride, and about 70% (w / v) sucrose at a pH of about 5.3. In some embodiments, the concentration of acetic acid in the excipient concentrated buffer may be about 2.0 mM to about 14.0 mM, about 6.0 mM to about 12.0 mM, about 8.0 mM to about 10.0 mM, about 2.0 mM to about 5.3 mM, or about 5.0 mM to about 6.0 mM. , about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5.0 mM, about 5.5 mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11.0 mM, about 11.5 mM, about 12.0 mM, about 12.5 mM, about 13.0 mM, about 13.5 mM, or about 14.0 mM. In some embodiments, the concentration of arginine hydrochloride in the excipient concentrated buffer is about 200 mM to about 500 mM, about 250 mM to about 450 mM, about 300 mM to about 400 mM, about The sucrose concentration in the excipient-concentrated buffer solution can be about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM. In some embodiments, the sucrose concentration in the excipient-concentrated buffer solution can be 50% to 90%, about 55% to 85%, about 60% to 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-concentrated buffer solution can be about 4.0 to about 6.5, about 4.5 to about 6.0, about 5.0 to 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.

[0460] After dilution with dilution buffer and combination with excipient concentration buffer, the adjusted FCP may be filtered and mixed into a single-use bag. The sample may be referred to as filtered drug substance (DS). The filtered DS may be further diluted to a target concentration with FDS dilution buffer and combined with excipient buffer solution to achieve the final concentration and composition.

[0461] The target concentration can be about 200.0 mg / mL. In some embodiments, the target concentration is about 160.0 mg / mL to about 240.0 mg / mL, about 180.0 mg / mL to about 220.0 mg / mL, about 190.0 mg / mL to about 210.0 mg / mL, about 195.0 mg / mL to about 205.0 mg / mL, about 199.0 mg / mL to about 201.0 mg / mL, about 199.5 mg / mL to about 200.5 mg / mL, about 199.9 mg / mL to about 200.1 mg / mL, about 160.0 mg / mL, about 165.0 mg / mL, about 170.0 mg / mL, or about 175.0 mg / mL. L, 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.

[0462] The FDS dilution buffer may comprise about 10 mM sodium acetate, about 25 mM alginate 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 can be about 2.0 mM to about 14.0 mM, about 6.0 mM to about 12.0 mM, about 8.0 mM to 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 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11.0 mM, about 11.5 mM, about 12.0 mM, about 12.5 mM, about 13.0 mM, about 13.5 mM, or about 14.0 mM. In some embodiments, the concentration of arginine hydrochloride in the FDS dilution buffer can be about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to 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 can be about 1% to 10%, about 2% to 9%, about 3% to 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 can be about 4.0 to about 6.5, about 4.5 to about 6.0, about 5.0 to 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.

[0463] The composition of the excipient buffer solution may be selected depending on the target concentration and composition of the final drug substance (FDS).

[0464] An excipient buffer solution for a target FDS concentration of 150 mg / mL can 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 at a pH of about 6.7. In some embodiments, the concentration of sodium acetate in the FDS excipient buffer solution (for a target FDS concentration of 150 mg / mL) can be about 5 mM to about 35 mM, about 10 mM to about 30 mM, about 15 mM to 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 (for a target FDS concentration of 150 mg / mL) can be about 60 mM to about 100 mM, about 65 mM to about 95 mM, about 70 mM to about 90 mM, about 75 mM to 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 (for a target FDS concentration of 150 mg / mL) can be about 10 mM to about 40 mM, about 15 mM to about 35 mM, about 20 mM to 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 (for a target FDS concentration of 150 mg / mL) can be about 1% to 10%, about 2% to 9%, about 3% to 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 (for a target FDS concentration of 150 mg / mL) can be about 0.1% to 1.6%, about 0.3% to 1.3%, about 0.6% to 1.1%, about 0.8% to 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 can be (for a target FDS concentration of 150 mg / mL) about 5.0 to about 8.0, about 5.5 to about 7.5, between about 6.0 to about 7.0, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5.

[0465] An excipient buffer solution for a target FDS concentration of 175 mg / mL can 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% polysorbate 80 at a pH of about 7.0. In some embodiments, the concentration of sodium acetate in the FDS excipient buffer solution (for a target FDS concentration of 175 mg / mL) can be about 15 mM to about 45 mM, about 20 mM to about 40 mM, about 25 mM to 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 can be about 120 mM to about 200 mM, about 130 mM to about 190 mM, about 140 mM to about 180 mM, about 150 mM to about 170 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, or about 180 mM (for a target FDS concentration of 175 mg / mL). In some embodiments, the concentration of L-arginine hydrochloride in the FDS excipient buffer solution (for a target FDS concentration of 175 mg / mL) can be about 125 mM to about 325 mM, about 150 mM to about 300 mM, about 175 mM to about 275 mM, about 200 mM to 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 (for a target FDS concentration of 175 mg / mL) can be about 1% to 10%, about 2% to 9%, about 3% to 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 can be about 0.8% to 2.4%, about 1.0% to 2.2%, about 1.2% to 2.0%, about 1.4% to 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% (for a target FDS concentration of 175 mg / mL).In some embodiments, the pH in the FDS excipient buffer solution can be about 5.5 to about 8.5, about 6.0 to about 8.0, about 6.5 to about 7.5, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0 (for a target FDS concentration of 175 mg / mL). [Example]

[0466] Example 1. Insulin replacement Total insulin concentrations (0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L) were assessed in combination with insulin schedules (total on day 4; split on days 2, 4; split on days 2, 4, 6, 8).

[0467] A modified seed train using normal operating conditions in the flask and bioreactor was used to inoculate the custom D-optimal design as shown in the table below (Table 9). A fed-batch process (Table 10) was run for 15 days in a 2 L bioreactor in CDM1B with taurine and sodium phosphate. Table 9 (Insulin replacement experiment conditions) [Table 9] Table 10 (Insulin-supplemented 2 L bioreactor conditions) [Table 10]

[0468] Bioreactor samples were measured for cell count and metabolites on a Nova Flex analyzer. Upon completion of production, samples were analyzed for titer analysis. The production period was 15 days, with bioreactor samples on day 13 analyzed for product quality analysis.

[0469] Increasing insulin resulted in higher productivity (Figure 2), higher viability (Figure 3), and reduced total ammonia (Figure 4) in some of the bioreactors. Higher inoculation densities also affected titer curve parameters, resulting in lower ammonia and viability.

[0470] From the JMP optimization (Figure 5), it was determined that the insulin concentration should be increased to 15 mg / L. Providing insulin on days 2 and 4 was preferred to maximize titers. As a result, an insulin feeding strategy of 7.5 mg / L on days 2 and 4 was found to be optimal.

[0471] Product quality was monitored periodically throughout media and feed development. Bioreactor samples taken on day 13 were submitted for product quality analysis. Galactosylation and fucosylation did not affect process variables. The impact of insulin feeding schedule was evaluated with respect to aggregates (Figure 6), non-reduced purity (Figure 7), and NGHC (Figure 8). The impact of insulin feeding schedule on these product quality attributes was minor and within development targets. Example 2. Tyrosine supplementation

[0472] During the ramp-up of the productivity process, it was noted that some amino acids were depleted. Consequently, additional tyrosine feeding was investigated to address the depletion.

[0473] A modified seed train using normal operating conditions in flasks was used to inoculate custom D-optimized flasks as shown in the table below (Table 11). The fed-batch process was run for 15 days in 250 mL flasks containing cell culture medium containing sodium phosphate and taurine. On production day 3, tyrosine was supplemented. The tyrosine concentration varied from 1.8 g / L to 2.2 g / L according to the experimental design. Table 11 [Table 11]

[0474] Feeding producing cells additional tyrosine resulted in ammonia reduction and had minimal impact on productivity, as shown in Figure 9. Example 3. Phosphate Redistribution

[0475] Phosphate is essential for energy transfer in cells and for the synthesis of nucleic acids and phospholipids. Phosphate depletion can lead to decreased cell viability and the accumulation of unwanted cellular metabolic by-products. Initial small-scale model development experiments showed potential phosphate depletion by day 2. Therefore, phosphate redistribution was studied, including increasing the basal medium concentration.

[0476] Phosphate feeding schedules were evaluated in the production flask model. Avoiding depletion of important nutrients, including phosphate, is fundamental and necessary before initiating more complex media and feed studies.

[0477] The modified seed train was used to inoculate flasks and production flask experiments utilizing a seed train bioreactor. A fed-batch process was run with the selected cell line in a flask containing starting production medium containing taurine. Depending on the experimental design, sodium phosphate levels in the starting medium were varied. Sodium phosphate was also varied in the four feeds during production (n=2 for each condition, see Table 12). Table 12 [Table 12]

[0478] Daily samples were analyzed for cell count on a Nova CDV, for metabolites on a Nova BP400, and for osmolality on an Advanced Instruments Model 2020 Osmometer. Samples were also subjected to titer analysis.

[0479] The phosphate redistribution strategy resulted in a slight increase in final titer (Figure 10). Different feeding paradigms altered the shape of the phosphate concentration curve, with three conditions approaching depletion at various production days (Figure 11). Conditions with early phosphate increases in the batch and continuous phosphate feeding (267, 525, 525, 250, 250 mg / L) until the end of the batch did not deplete phosphate during production; this feeding strategy was implemented for the upstream process. Example 4. Production Bioreactor Conditions

[0480] Since the majority of the protein is produced in this unit operation, the cell culture parameters in the production bioreactor are expected to have the most impact on both product quality and process productivity.

[0481] The seed train-derived culture was inoculated into two N-3 10 L stirred-tank bioreactors. After reaching N-3 final density, three N-2 10 L stirred-tank bioreactors were inoculated. After reaching N-2 final density, four N-1 10 L stirred-tank bioreactors were inoculated. Appropriate split ratios were used throughout the seed train expansion. A 2 L fed-batch production bioreactor was inoculated with three specified final N-1 density targets. Three of the four N-1 reactors were utilized to inoculate the production bioreactor. In addition to the inoculation-related variable of density, temperature and upper pH limits were set and the lower pH limit was held constant according to the experimental design.

[0482] Daily samples were analyzed on the Nova FLEX for cell count and metabolites. Upon completion of the production batch, samples from days 10, 12, and / or 13 were analyzed for titer analysis and quality testing. Sixteen samples from days 10, 12, and / or 13 were also analyzed for sequence variants.

[0483] Harvest date was determined to have a significant impact on preliminary general quality attributes, process characteristics, and sequence variants (titer, total fucosylation, total galactosylation, viability, total Pro-Ala SV, Pro-Ala Site P12). Harvest date is a timing-dependent upstream process parameter for which an operating range is defined, taking into account manufacturing facility capacity and flexibility. Monte Carlo simulations were performed to determine the range combinations. Based on the JMP desirability maximization algorithm, the optimal profiler determined day 10.0 as the optimal setpoint.

[0484] Monte Carlo simulations were performed for N-1 densities to characterize the failure rates for process and quality attributes for the combined tested process range. An ideal process range was then selected based on achieving the most desirable manufacturing process with a focus on minimizing failure rates (target failure rate <100 ppm, 0.01%). Failures primarily occurred in meeting Cys-Tyr substitution, SDS LMW, SDS purity, SDS purity (HC+LC+NGHC), SEC HMW aggregation dimer, and SEC HMW aggregation higher order targets. Increasing the upper N-1 density limit had the greatest impact on Cys-Tyr substitution (2.0% increase) and minimal impact on quality attributes. The N-1 density range was chosen from 4.35 to 5.45 x 10, balancing quality attribute response and manufacturing flexibility. 5 was set to cells / mL.

[0485] Increasing the upper harvest date limit increased NR SDS LMV failure by 70% and reduced SEC HMW aggregate high order by 22%. The NR SDS LMW increase, due to the reduction in trisulfides, simultaneously reduced SDS LMW and harvest length. Due to this inconsistency and to increase ease of manufacturing, the harvest date range was set at 10-11 days. A final harvest date range and N-1 density range of 5 standard deviations were selected for the final process to balance tighter control (lower failure rate) and manufacturing flexibility. A summary of the simulation conditions can be seen in Table 13 (Production Simulation Condition Summary). Table 13 (Summary of production simulation conditions) [Table 13] Collection outline

[0486] The harvesting process can begin with a separation step after recombinant protein production using the upstream production methods described above and / or by alternative production methods conventional in the art. 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, particulate debris from either host cells or lysed cells (e.g., resulting from homogenization) can be removed by a variety of means, including, but not limited to, centrifugation or ultrafiltration. If the protein of interest is secreted into the culture medium, supernatants from such expression systems can first be concentrated using commercially available protein concentration filters, for example, Amicon™ or Millipore Pellicon™ ultrafiltration devices. In one embodiment, the protein of interest can be harvested by centrifugation, followed by depth filtration, and then affinity capture chromatography.

[0487] A variety of different purification techniques are contemplated within the scope of the present invention, including, but not limited to, affinity, ion exchange, mixed-mode, size exclusion, and hydrophobic interaction chromatography, either singly or in combination. These chromatography steps separate the protein mixture of a biological sample based on its charge, degree of hydrophobicity, or size, or a combination thereof, depending on the specific type of separation. As noted above, several different chromatography resins are available for each technique, allowing the purification scheme to be precisely tailored to the specific proteins involved. Each separation method results in physical separation, with proteins traversing the column at different rates, increasing as they pass further through the column or selectively adhere to the separation medium. Proteins are then (i) differentially eluted using an appropriate elution buffer and / or (ii) optionally collected from the flow-through fractions obtained from the column used, following washing of the column with an appropriate equilibration buffer. In some cases, a protein of interest is separated from impurities (HCPs, protein variants, etc.) when the impurities preferentially adhere to the column and the protein of interest adheres less, e.g., the protein of interest does not adsorb to the solid phase of a particular column and therefore flows through the column. In some cases, impurities are separated from the protein of interest when they cannot adsorb to the column and therefore flows through the column.

[0488] The harvesting process can begin with a separation step after the recombinant protein has been produced using the upstream production method described above and / or by another production method conventional in the art. Once a clarified solution or mixture containing the protein of interest, e.g., dupilumab, is obtained, separation of the protein of interest from process-related impurities (e.g., other proteins produced by the cells (such as HCPs) and product-related substances, e.g., acidic or basic variants) is performed. A combination of one or more different production techniques may be used, including affinity, ion exchange (e.g., CEX, AEX), mixed-mode (MM), and / or hydrophobic interaction chromatography. Such a production step separates a mixture of components within a biological sample based, for example, on their charge, degree of hydrophobicity, and / or apparent size. Many chromatography resins are commercially available for each of the chromatography techniques mentioned herein, allowing the method scheme to be precisely tailored to the specific protein involved. Each separation method allows proteins to traverse the column at different rates, achieving a physical separation that increases as they pass further through the column or are selectively adsorbed to the separation resin (or medium). The proteins can then be differentially collected. In some cases, a protein of interest is separated from components of a biological sample when other components are specifically adsorbed to the resin of the column, while the protein of interest is not.

[0489] Column loading and wash steps can be controlled by in-line, at-line, or off-line measurement of product-related impurity / substance levels in either the column effluent, or the collection pool, or both, to achieve a particular 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 buffers or other solutions to achieve the required distribution for improved separation efficiency and / or yield. Pre-harvest processing

[0490] In certain exemplary embodiments, the harvest process may include an initial step that slows the enzymatic process to reduce aggregate formation. Primary recovery may also be combined with the use of pretreatment. Pretreatment is designed to improve the harvest step through the use of additives that aid in cell removal and reduce process-related impurity levels, thereby lowering the load on downstream chromatography steps. Pretreatments include flocculation or precipitation, both of which promote the association or aggregation of smaller particulates to form larger solids that can be more efficiently removed. Non-limiting examples of these pretreatments include polymeric flocculants, chitosan, poly(ethyleneimine), and cationic salts, such as calcium phosphate.

[0491] Examples of pre-harvest treatments that can be used in the present invention are described in more detail in the Examples below. In one aspect, this step can be carried out by a process of cooling the bioreactor to about (±3°C) 13.5°C, 14.5°C, 15.5°C, 16.5°C, 17.5°C, 18.5°C, 19.5°C, 20.5°C, 21.5°C, 22.5°C, or 23.5°C. Maintaining a narrow temperature range has also been shown to limit changes in impurity solubility or polish filter binding during advanced steps. Example 5: Chitosan flocculation pretreatment

[0492] In one example, chitosan was dissolved in 1% (w / v) acetic acid, while a 125 mM stock solution of calcium phosphate was generated from equal parts 500 mM sodium phosphate and 800 mM calcium chloride. Cell culture media for polyethyleneimine (PEI) and chitosan originated from BRX161-10128, while material for calcium phosphate originated from two development bioreactors, TD29-10437 and TD32-10438. Pools of cell culture media were adjusted to three different pH values ​​using acetic acid: native pH, pH 6.5, and midpoint pH. From these three different pools, aliquots of each pH were adjusted to three different conductivity ranges: native, 1.5x native, and 0.5x native, by adding NaCl or diluting with RODI.

[0493] A summary of the test samples can be seen in Tables 14, 15, and 16 below. Table 14 (Summary of Flocculent Central Composite Design (CCD) of Design of Experiments (DoE) studies performed on PEI, CF = Clearance Factor (loading) HCP / Pool HCP )) [Table 14] Table 15 (Summary of flocculation CCD DoE studies p...

Claims

1. At least 2.5x10 5 A method of producing dupilumab comprising culturing cells at an initial viable cell density (VCD) in a seed train adjusted to be cells / mL.

2. The seed train comprises cell cultures in N-5 to N-1 vessels or bioreactors, and the initial VCD is 3.5x10 in each vessel or bioreactor. 5 ~5.43x10 5 The method of claim 1, adjusted to cells / mL.

3. The final dupilumab titer is greater than or equal to 2.5x10 initial VCD in the N-5 to N-1 vessels or bioreactors. 5 3. The method of claim 2, wherein the titer is about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 1%, 17%, 18%, 19%, or 20% higher than the titer produced from a cell culture that is less than 100 cells / mL.

4. 2. The method of claim 1, wherein the initial VCD is about 1.3x, 1.4x, 1.5x, 1.6., 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, or 3.0x larger than an alternative initial VCD in a standard seed train.

5. 4. The method of claim 3, wherein the increased final dupilumab titer is independent of the final VCD in the N-1 seed train vessel or bioreactor.

6. 2.5 x 10 initial VCD vessels or bioreactors in the seed train 5 3. The method of claim 2, wherein the peak lactate observed in the final production vessel is not substantially different compared to the final production vessel which is less than cells / mL.

7. 7. The method of claim 6, wherein the seed train produces an increase in final titer (g / L) of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 1%, 17%, 18%, 19%, or 20%.

8. Initial VCD during seed train is 3.5x10 5 ~5.43x10 5 8. The method of claim 7, adjusted to cells / mL.

9. 1. A method of culturing cells comprising: a) measuring a first capacitance value of a first cell culture using at least one online capacitance probe; b) measuring a first viable cell density value of said first cell culture using at least one offline assay; c) correlating the first capacitance value with the first viable cell density value to determine a correlation equation; d) determining a second capacitance value of the second cell culture using an online capacitance probe; and e) predicting at least one second viable cell density value of the second cell culture using the second capacitance value and the correlation equation; and f) adjusting a working volume or viable cell density (VCD) based on said second viable cell density value to culture said cells. The method comprising:

10. 10. The method of claim 9, wherein the cells are from the same cell line as the cell line used to obtain the correlation equation.

11. 10. The method of claim 9, wherein the correlation equation is generated using multivariate data analysis or linear regression.

12. 1. A method of producing dupilumab, comprising: (a) maintaining the initial viable cell density (VCD) in the seed train in a vessel or bioreactor at at least 2.5 x 10 5 adjusting the concentration to cells / mL and culturing the cells; (b) Viable cell density (i) applying an electric field to the cells cultured in a vessel or bioreactor; (ii) measuring capacitance; and (iii) Correlating capacitance to viable cell density; and measuring by (c) adjusting the initial VCD in each seed train vessel or bioreactor; (d) producing dupilumab. The method comprising:

13. The initial VCD in each vessel or bioreactor in the N-5 to N-1 seed train is 3.5x10 5 ~5.43x10 5 The method of claim 12, adjusted to cells / mL.

14. The final dupilumab titer was 2.5x10 compared to the initial VCD in the seed train. 5 13. The method of claim 12, wherein the titer is about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 1%, 17%, 18%, 19%, or 20% higher than the titer produced from a cell culture that is less than 100 cells / mL.

15. 14. The method of claim 13, wherein the initial VCD is about 1.3x, 1.4x, 1.5x, 1.6., 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, or 3.0x larger than an alternative initial VCD in a standard seed train.

16. 16. The method of claim 15, wherein the increased final dupilumab titer is independent of the final VCD in the N-5 to N-1 vessel or bioreactor.

17. The initial VCD in each N-5 to N-1 vessel or bioreactor is 2.5 x 10 5 13. The method of claim 12, wherein the peak lactate observed in the final production vessel is not substantially different compared to the final production vessel or bioreactor which is less than cells / mL.

18. 18. The method of claim 17, wherein the seed train produces an increase in final titer (g / L) of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 1%, 17%, 18%, 19%, or 20%.

19. 1. A method for producing an anti-IL-4Rα antibody or antigen-binding fragment thereof, comprising: a) culturing in a vessel or bioreactor a cell capable of expressing an anti-IL-4Rα antibody or an antigen-binding fragment thereof; b) Initial viable cell density (VCD) of 2.5x10 5 Adjust to cells / mL or more. c) measuring a first property of the first cell culture using at least one online sensor; and d) measuring a second property of the first cell culture using at least one offline assay; and e) correlating the measurements of the first property of the first cell culture with the measurements of the second property of the first cell culture to determine a correlation equation; f) measuring the first property of the second cell culture using an online sensor; and g) predicting at least one measurement of the second property of the second cell culture using the measurement of the first property of the second cell culture and the correlation equation; and h) transferring the cells to another vessel or bioreactor based on the at least one predicted measurement value of the second property of the second cell culture; and i) repeating steps b) through h) along the seed train; The method comprising:

20. 20. The method of claim 19, wherein the cells are derived from the same cell culture as the first cell culture.

21. 20. The method of claim 19, wherein the cells are from a cell culture different from the first cell culture.

22. 20. The method of claim 19, wherein the correlation equation is obtained using more than one cell line.

23. 20. The method of claim 19, wherein more than one measurement of the first property of the first cell culture is performed.

24. 20. The method of claim 19, wherein more than one measurement of the second property of the first cell culture is performed.

25. 20. The method of claim 19, wherein at least about 50 percent of the variability in the measurement of the second characteristic of the first cell culture is due to the variability in the measurement of the first characteristic of the first cell culture.

26. 20. The method of claim 19, wherein the correlation equation is generated using multivariate data analysis or linear regression.

27. 20. The method of claim 19, wherein the initial VCD is about 1.3x, 1.4x, 1.5x, 1.6., 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, or 3.0x larger than an alternative initial VCD in a standard seed train.

28. The initial VCD in N-5 to N-1 containers is 2.5x10 5 20. The method of claim 19, wherein the peak lactate observed in the final production vessel or bioreactor is not substantially different compared to the final production vessel which is less than cells / mL.

29. 30. The method of claim 28, wherein the seed train produces an increase in final titer (g / L) of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

30. 30. The method of claim 28, wherein the initial VCD is about 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, or 3.0x higher than an alternative initial VCD in a standard seed train.

31. 20. The method of claim 19, wherein the cell is a CHO cell.

32. The method of claim 19, wherein the anti-IL-4Rα antibody is dupilumab.

33. A method for treating a patient having a type 2 inflammatory disease, comprising administering to the patient an anti-IL-4Rα antibody, antigen-binding fragment thereof, or dupilumab produced according to the method of any one of claims 1-8 or 12-32.

34. 34. The method of claim 33, wherein the type 2 inflammatory disease is atopic dermatitis, moderate to severe atopic dermatitis, asthma, moderate to severe asthma, allergic rhinitis, chronic rhinosinusitis with nasal polyps, eosinophilic esophagitis, chronic obstructive pulmonary disease, chronic spontaneous urticaria, prurigo nodularis, allergic fungal rhinosinusitis, chronic rhinosinusitis without nasal polyps, allergy, grass allergy, peanut allergy, dairy allergy, bullous pemphigoid, atopic dermatitis of the hands and feet, cold-induced urticaria, chronic induced urticaria, ulcerative colitis, chronic pruritus of unknown cause, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, bronchiectasis, or alopecia areata.

35. A method for treating a disease or disorder associated with IL-4R activity, comprising administering to a patient an anti-IL-4Rα antibody, an antigen-binding fragment thereof, or dupilumab produced according to the method of any one of claims 1 to 8 or 12 to 32.

36. 36. The method of claim 35, wherein the disease or disorder associated with IL-4R activity is atopic dermatitis, moderate to severe atopic dermatitis, asthma, moderate to severe asthma, allergic rhinitis, chronic rhinosinusitis with nasal polyps, eosinophilic esophagitis, chronic obstructive pulmonary disease, chronic spontaneous urticaria, prurigo nodularis, allergic fungal rhinosinusitis, chronic rhinosinusitis without nasal polyps, allergy, grass allergy, peanut allergy, dairy allergy, bullous pemphigoid, atopic dermatitis of the hands and feet, cold-induced urticaria, chronic induced urticaria, ulcerative colitis, chronic pruritus of unknown etiology, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, bronchiectasis, or alopecia areata.

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