Cell culture method for antibody production

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0003] Sequence Listing

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

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

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

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

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

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

[0010] To enable processing of higher titer drug products, improved methods and systems for manufacturing, collecting, and purifying high-titer antibody drug products have been developed. For example, increased drug product titer and drug loading can exceed the capacity of certain chromatographic purification processes and alter viscosity, which adversely affects the transfer of drug product along different chromatographic steps. Therefore, improved methods are needed that address not only individual purification steps but also the entire manufacturing process. Summary of the Invention

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

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

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

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

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

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

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

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

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

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

[0021] In one embodiment, the PS medium is serum-free and comprises spermidine at a concentration between 10 μM and 900 μM. In one aspect, spermidine is present at a concentration of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, In one embodiment, the PS medium contains 57 mg / L ± 8.55 mg / L of spermine·4HCl and ≥ 15 mg / L ± 2.25 mg / L of spermidine·HCl. In some embodiments, the medium contains ≤ 7.5 g / L of hydrolyzate. In some embodiments, the medium contains ≤ 16 g / L of hydrolyzate. In some embodiments, the PS medium does not contain any hydrolyzate.

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

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

[0024] In some embodiments, the culture medium comprises one or more fatty acids. In one aspect, the culture medium comprises a mixture of fatty acids (or fatty acid derivatives) and alpha tocopherol. In one aspect, the fatty 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, capric acid, dodecanoic acid, caproic acid, lignoceric acid, myristic acid, and caprylic acid, and may also include lipoic acid derived from the fatty acid.

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

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

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

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

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

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

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

[0032] In some embodiments, the cell culture medium: (1) is serum-free; (2) comprises a hydrolysate; (3) comprises one or more polyamines, including at least ornithine or spermine; (4) comprises at least about 40 mM or at least about 70 mM of a mixture of amino acids, including at least one of the following: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof; (5) comprises a mixture of tocopherols and fatty acids; (6) comprises a mixture of nucleosides, including at least one of the following: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; (7) comprises a salt of at least one of the following: calcium, magnesium, and phosphate; and (8) comprises at least one of the following point-of-use additives: NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4 EDTA, trisodium citrate, and combinations thereof.

[0033] In some embodiments, the cell culture medium: (1) is serum-free; (2) contains one or more polyamines, including at least ornithine or spermine; (3) contains at least about 40 mM or at least about 70 mM of a mixture of amino acids, including at least one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof; (4) contains a mixture of tocopherols and fatty acids; (5) contains a mixture of nucleosides, including at least one of adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; (6) contains salts, including at least one of calcium salts, magnesium salts, phosphate salts, and combinations thereof; and (7) contains at least one of the following point-of-use supplements: NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4 EDTA, trisodium citrate, and combinations thereof.

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

[0035] In some embodiments, the cell culture medium: (1) does not contain hydrolysis products; (2) contains ornithine at a concentration of at least 90 μM ± 14 μM; 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 to be within the scope of the present invention.

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

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

[0038] In some embodiments, the protein of interest is produced by the following steps: (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 carrying the nucleic acid sequence; (3) culturing the selected cells in a serum-free cell culture medium comprising: (a) 0.09±0.014, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM ornithine; (b) optionally, 0.20±0.03 mM, 0.35±0.06, or 0.714±0.11 mM putrescine; (c) at least 40 mM or at least 70 mM of a mixture of amino acids. (d) a mixture of tocopherols and fatty acids; (e) a mixture of nucleosides comprising one or more of adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; and (f) a salt comprising one or more of a calcium salt, a magnesium salt, and a phosphate salt; and (4) expressing a protein of interest in CHO cells, wherein the protein of interest is secreted into the culture medium. In some embodiments, the serum-free cell culture medium may comprise ≤7.5 g / L of hydrolysate; or in other embodiments, the serum-free cell culture medium may comprise no hydrolysate at all.

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

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

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

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

[0043] In some embodiments, the sparging rate can be used to alter the pCO2 level within the cell culture to modulate the acidic and basic variants of cultured cells expressing dupilumab.

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

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

[0046] Thus, the present disclosure provides an improved system and method for reducing signal noise in electrochemical probes that reduces or eliminates false positives attributable to bubble interference. Additionally, the present disclosure provides a system and method for improving the accuracy of optical probes for measuring dissolved gases in large-scale containers and bioreactors that require less maintenance than conventional electrochemical probes and are less susceptible to bubble interference. As discussed in more detail below, containers and bioreactors with electrochemical and optical probes are developed with improved data processing methods to optimize the accuracy of measurements while minimizing maintenance by reducing or eliminating unwanted excursion events outside the normal operating range, which requires lengthy quality studies in the strictly regulated biopharmaceutical industry.

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

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

[0049] To collect and purify the increased protein product, new and improved methods were developed that not only accommodated the increased protein yield but also improved the overall yield percentage. These methods included, for example, optimized collection pretreatment, Protein A chromatography, viral inactivation, anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, viral retention filtration, concentration and diafiltration, and drug substance conditioning, which are further described below.

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

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

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

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

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

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

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

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

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

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

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

[0061] In one aspect, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises three heavy chain 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.

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

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

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

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

[0066] Figure 2 Graphs show the effect of insulin supplementation on the titer profile of dupilumab according to exemplary embodiments.

[0067] Figure 3 Graphs show the effect of insulin supplementation on the viability of dupilumab-expressing cells according to exemplary embodiments.

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

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

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

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

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

[0073] Figure 9 Shown are the effects of tyrosine supplementation on titer and ammonia levels in cell cultures expressing dupilumab, according to exemplary embodiments.

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

[0075] Figure 11Shown are phosphate redistribution profiles for days 0, 2, 4, 6, and 8, according to an exemplary embodiment.

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

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

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

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

[0080] Figure 14 Shown are the initial and final viable cell densities (VCD) of an average standard seed-expansion culture and an optimized seed-expansion culture with an increased initial VCD compared to the standard seed-expansion culture, according to exemplary embodiments.

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

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

[0083] Figure 17 Shown are bivariate linear regressions of online capacitance measurements and offline VCD measurements for cell line A (CLA) during seed expansion culture according to an exemplary embodiment of the present invention.

[0084] Figure 18 Bivariate linear regression of online capacitance measurements and offline VCD measurements of CLA during the N-3, N-2, and N-1 seed expansion stages according to an exemplary embodiment of the present invention is shown.

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

[0086] Figure 20 Shown are bivariate linear regressions of online capacitance measurements and offline VCD measurements during seed expansion culture for cell line B (CLB) according to an exemplary embodiment of the present invention.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0100] Figure 33Shown are the HCP contents in affinity capture pools when various affinity wash buffers were used according to exemplary embodiments.

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

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

[0103] Figure 36 Observations from a model generated from a multivariate study of AEX risk factors and responses are shown, according to an exemplary embodiment.

[0104] Figure 37 Observations from a model generated from a multivariate study of CEX risk factors and responses are shown, according to an exemplary embodiment.

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

[0106] Figure 39 Observations from a model generated from a multivariate study of VRF risk factors and responses are shown, according to an exemplary embodiment.

[0107] Figure 40 Observations from a model generated from a multivariate study of UF / DF risk factors and responses are shown, according to an exemplary embodiment.

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

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

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

[0111] Figure 44 Shown are the HCP content of the unit operations for a 500 L confirmation batch compared to a 10,000 L process performance verification batch according to an exemplary embodiment.

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

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

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

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

[0116] Figure 49 Showing the Figures 46 to 48 The superposition comparison of the filtered signals.

[0117] Figure 50 Shown is the dissolved oxygen trend for Run 1 according to an exemplary embodiment (light line is electrochemistry, medium line is optical probe B, darkest line is optical probe A, dashed lines: set point and upper and lower normal operating range limits).

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

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

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

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

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

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

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

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

[0126] Figure 59 Shown are the pCO2 profiles (y-axis) of culture media with different sparging conditions (in mmHg) as a function of treatment time (days) according to an exemplary embodiment. A medium pCO2 was chosen as a midpoint control.

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

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

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

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

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

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

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

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

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

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

[0137] Description of terms

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

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

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

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

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

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

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

[0145] As used herein, the term "recombinant host cell" (or simply "host cell") includes cells into which a recombinant expression vector encoding a protein of interest is introduced. It should be understood that such terms are intended to refer not only to specific individual cells, but also to the progeny of such cells. Because certain modifications may occur in progeny due to mutations or environmental influences, such progeny may not actually be consistent with the parental cells, but are still included within the scope of the term "host cell" as used herein. In one embodiment, host cells include prokaryotes and eukaryotic cells selected from any kingdom of life. In one aspect, eukaryotic cells include protozoa, fungi, plants, and animal cells. In another aspect, host cells include eukaryotic cells, such as plant and / or animal cells. The cell may be a mammalian cell, a fish cell, an insect cell, an amphibian cell, or an avian cell. In a specific aspect, the host cell is a mammalian cell. A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and other repositories and commercial suppliers. Cells that can be used in the methods of the present invention include, but are not limited to, MK2.7 cells, PER-C6 cells, Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCCCL-61) (Chasin et al., 1986, 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. Pat. No. 5,721,121); monkey kidney cells (CV1, ATCC CCL-70); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651);HEK293 cells, Sp2 / 0 cells, 5L8 fusion tumor cells, Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkat cells, THP-1 cells, Sp2 / 0 cells, primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells) and established cell lines and strains thereof (e.g., human embryonic kidney cells (e.g., HEK293 cells, or HEK293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); mouse Sethroid cells (e.g., mouse embryonic kidney cells, e ... mouse embryonic kidney cells, e.g., mouse embryonic kidney cells, e.g., mouse embryonic kidney cells, e.g., mouse embryonic kidney cells, e.g., mouse embryonic kidney cells, e.g., mouse embryonic kidney cells, e.g., mouse embryonic kidney cells, e.g., mouse embryonic kidney cell) (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, LS180 cells, LS174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28VA13, 2RA cells, WISH cells, BS-CI cells, LLC-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-1, B1 cells or their derivatives), fibroblasts from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoids, tonsil, bone marrow and blood), spleen), and fibroblasts and fibroblast-like cell lines (e.g., TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, 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, KLN205 cells, McCoy cells, mouse L cells, line 2071 (mouse L) cells, line LM (mouse L) cells, L-MTK (mouse L) cells, NCTC strains 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, C; IICells and Jensen cells, or derivatives thereof), or any other cell type known to those skilled in the art. In some aspects, the cells used in the present invention are CHO cells, HEK293 cells, BHK cells, or derivatives thereof.

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

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

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

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

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

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

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

[0153] As used herein, the term "database" refers to a collection of protein sequences that may be present in a sample, for example, in the form of a FASTA-formatted file. Related protein sequences can be derived from cDNA sequences of the species being studied. Public databases that can be used to search for related protein sequences include databases hosted by, for example, Uniprot or Swiss-prot. Databases can be searched using tools referred to herein as "bioinformatics tools." Bioinformatics tools provide the ability to search uninterpreted MS / MS spectra relative to all possible sequences in the database 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).

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

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

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

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

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

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

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

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

[0162] For biologics, the implementation of robust and flexible upstream processes is desirable. Efficient upstream processes can lead to the desired production and scale-up of the protein of interest.

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

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

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

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

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

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

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

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

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

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

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

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

[0175] Exemplary anti-IL-4Rα antibodies

[0176] As used herein, dupilumab (IgG4 isotype) is a covalent heterotetramer composed of two disulfide-linked human heavy chains, each covalently linked to a human kappa light chain via a disulfide bond. Each heavy chain contains a serine to proline mutation at amino acid 233 in the hinge region of the Fc domain to reduce the tendency of the antibody to form half antibodies in solution. There is a single N-linked glycosylation site (Asn) in each heavy chain. 302 ), which is located within the CH2 domain of the Fc constant region of the molecule. The antibody based on the primary sequence (absence of N-linked glycosylation) has a molecular weight of 146,897.0 Da, taking into account the formation of 16 disulfide bonds and the removal of Lys from the C-terminus of each heavy chain. 452 The variable domains of the heavy and light chains combine to form the complementarity determining regions (CDRs) that bind dupilumab to its target, interleukin-4 receptor alpha (IL-4Rα). Figure 1 A schematic representation of dupilumab, including the location of N-linked glycosylation sites and disulfide bond structure, is presented in FIG.

[0177] The manufacturing process can be used to produce human antibodies or antigen-binding fragments thereof that specifically bind to an IL-4R (e.g., IL-4Rα) and comprise the 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 can comprise the three light chain CDRs (LCVR1, LCVR2, and LCVR3) 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 can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can 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 compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases can also be used to identify CDR sequences within antibodies.

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

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

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

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

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

[0183] Dupilumab HCVR amino acid sequence:

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

[0185] Dupilumab LCVR amino acid sequence:

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

[0187] Dupilumab HCDR1 amino acid sequence:

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

[0189] Dupilumab HCDR2 amino acid sequence:

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

[0191] Dupilumab HCDR3 amino acid sequence:

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

[0193] Dupilumab LCDR1 amino acid sequence:

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

[0195] Dupilumab LCDR2 amino acid sequence:

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

[0197] Dupilumab LCDR3 amino acid sequence:

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

[0199] Dupilumab HC amino acid sequence:

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

[0201] Dupilumab LC amino acid sequence:

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

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

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

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

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

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

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

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

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

[0211] Table 1

[0212]

[0213]

[0214]

[0215]

[0216]

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

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

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

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

[0221] Table 2

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

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

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

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

[0233] Table 3

[0234]

[0235]

[0236]

[0237]

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

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

[0240] Table 4

[0241]

[0242]

[0243]

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

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

[0246] Cell culture media and supplements

[0247] Unfortunately, removing serum from cell culture media and reducing or eliminating hydrolysates, while reducing batch-to-batch variability and facilitating downstream processing steps, can impair cell growth, reduce viability, and decrease protein expression. Consequently, chemically defined serum-free and low- or even hydrolysate-free culture media require additional components to improve cell growth and protein production. The cell culture media of the present invention can 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, and the like, depending on the needs of the cells being cultured or the desired cell culture parameters.

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

[0249] In one aspect, PS medium comprises a concentration (expressed in micromoles / liter) of at least about 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 540, 545, 550, 555, 560, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1000,

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

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

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

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

[0254] Table 5

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

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

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

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

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

[0260] In one embodiment, in addition to comprising ornithine or a combination of ornithine and putrescine, the culture medium also comprises a micromolar amount of fatty acids (or fatty acid derivatives) and tocopherols. In one embodiment, the fatty acids comprise 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, capric acid, dodecanoic acid, caproic acid, lignoceric acid, myristic acid, and caprylic acid. In one embodiment, the culture medium comprises tocopherols, linoleic acid, and thioctic acid.

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

[0262] In one embodiment, culture medium also includes taurine, hypotaurine or its combination. Taurine is present in many tissues of human and other mammalian species, such as brain, retina, myocardium, skeleton and smooth muscle, platelets and neutrophils. Taurine can contribute to osmotic pressure regulation, membrane stabilization and anti-inflammatory, and regulates mitochondrial protein synthesis (Jong et al., 2010, Journal of Biomedical Science (Journal of Biomedical Science) 17 (Supplement 1) via the enhanced electron transport chain activity that avoids superoxide production: S25; Jong et al., 2012, amino acids (Amino Acids) 42: 2223-2232sw). In one aspect, serum-free cell culture medium can include about 0.1mM to about 10mM taurine, about 0.1mM to about 1mM taurine, about 0.4mM to about 8mM taurine, about 1mM to about 6mM taurine, about 2mM to about 5mM taurine or about 3 to about 4mM taurine. It has also been found that taurine increases the specific productivity of cells and enables those cells to produce less ammonia byproduct, thereby improving the production of recombinant proteins. See U.S. Patent No. 10,927,342, which is incorporated by reference in its entirety.

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

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

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

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

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

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

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

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

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

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

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

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

[0275] In one aspect, a fed-batch production method for preparing dupilumab comprises culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding dupilumab at 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.

[0276] In one aspect, a fed-batch production method for preparing dupilumab comprises culturing Chinese hamster ovary (CHO) cells containing a nucleic acid encoding dupilumab at 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0291] Applicants contemplate that any one or more or combinations of a variety of basal media may be used in the practice of the present invention to which ornithine or a combination of ornithine and putrescine may be added. Basal media are generally known in the art and include, among others, 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-12K medium, Dulbecco's medium, Dulbecco's modified Eagle's medium (Proc. Nat'l. Acad. Sci. USA, 1952, Aug; 38(8):747-752), 1:1 DMEM / Ham's F12, Trowell's T8, A2 medium (Holmes and Wolf, Biophys. Biochem. Cytol., 1961, 10: 389-401), Waymouth medium (Davidson and Waymouth, Biochem. J., 1945, 39 (2): 188-199), Williams E medium (Williams et al., Exp. Cell Res., 1971, 69: 105 et seq.), RPMI1640 (Moore et al., J. Amer. Med. Assoc., 1967, 199: 519-524), MCDB 104 / 110 medium (Bettger et al., Proceedings of the National Academy of Sciences of the United States of America, 1981, 78 (9): 5588-5592), Ventrex HL-1 medium, albumin-globulin medium (Orr et al., Appl. Microbiol.), 1973, 25(1):4954), RPMI-1640 medium, RPMI-1641 medium, Iscove's Modified Dulbecco's Medium, McCoy 5A medium, Leibovitz L-15 medium, and serum-free medium, such as EX-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 Re 30,985), transferrin-fatty acid medium (Baker, 1982, US 4,560,655), transferrin-EGF medium (Hasegawa, 1982, US 4,615,977; Chessebeuf, 1984, US 4,786,599) and other media substitutes (see Inlow, US 6,048,728; Drapeau, US 7,294,484; Mather, US 5,122,469; Furukawa, US 5,976,833; Chen, US 6,180,401; Chen, US 5,856,179; Etcheverry, US 5,705,364; Etcheverry, US 7,666,416; Ryll, US 6,528,286; Singh, US 6,924,124; Luan, US 7,429,491, etc.).

[0292] In certain embodiments, the culture medium is chemically defined and comprises, in addition to ornithine or a combination of ornithine and putrescine: CaCl2 2H2O; HEPES buffer, KCL; MgSO4; NaCl; Na2HPO4 or other phosphates; pyruvate; L-alanine; L-arginine HCl; L-asparagine HCl 0; L-aspartic acid; L-cysteine ​​HCl 0; L-glutamic acid; glycine; L-histidine HCl H2O; L-isoleucine; L-leucine; L-lysine HCl; L-methionine; L-ornithine HCl; L-phenylalanine; L-proline; L-serine; L-threonine; L-tryptophan; L-tyrosine 2Na2H2O; L-valine; D-biotin; choline chloride; folic acid; inositol; niacinamide; pyridoxine HCl; D-pantothenic acid; riboflavin; thiamine HCl; vitamin B12; para-aminobenzoic acid; ethanolamine HCl; poloxamer 188; DL-a-tocopherol phosphate; linoleic acid; Na2SeO3; lipoic acid; and glucose; and optionally adenosine; guanosine; cytidine; uridine; thymidine; and hypoxanthine 2Na.

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

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

[0295] The present disclosure provides a cell culture comprising a cell line expressing a protein of interest in an OS culture medium as described above. In one embodiment, the cell culture comprises insulin, which can be added to the culture medium as a point-of-use ingredient or can be included in the culture medium formulation. In one embodiment, the cell line comprises cells capable of producing a biotherapeutic protein. Examples of cell lines routinely used for the production of protein biotherapeutics include, among others, 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 embryonic cells. In one embodiment, the cell line is a CHO cell line, or one or more of several specific CHO cell variants optimized for large-scale protein production, such as CHO-K1.

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

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

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

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

[0300] Chelating agents are also well known in the art of cell culture and protein production. Tetrasodium EDTA dihydrate and citrate are two common chelating agents used in the art, but other chelating agents can be used in the practice of the present invention. In one embodiment, the point-of-use additive chelating agent is tetrasodium EDTA dihydrate. In another embodiment, the point-of-use additive chelating agent is a citrate, such as Na3C6H5O7.

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

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

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

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

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

[0306] In some embodiments, the doubling rate of viable 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-fold greater than the doubling rate of mammalian cells cultured in non-PS medium. In some embodiments, the doubling rate of viable mammalian cells in PS medium is about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% greater than the doubling rate of mammalian cells in non-PS medium.

[0307] 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 cells cultured in PS medium is at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, or at least 25% shorter than the doubling time of actively cycling cells cultured in non-PS medium.

[0308] Protein production

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

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

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

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

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

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

[0315] In one embodiment, the cell is a CHO cell that ectopically expresses the protein. In one embodiment, the protein comprises an immunoglobulin heavy chain region, such as a CH1, CH2, or CH3 region. In one embodiment, the protein comprises 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).

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

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

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

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

[0320] Seed expansion culture optimization and measurement

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0337] In one embodiment, the present disclosure provides a method for culturing cells by: measuring a first property of the cell culture using an inline 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 used to culture the cells based on the at least one predicted measurement of the second property of the cell culture.

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

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

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

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

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

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

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

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

[0346] In one embodiment, a bioanalyzer system can be used to perform bioanalyzer measurements of cell culture samples. The bioanalyzer system can include at least one 96-well plate, a syringe, a 24-position external sample tray, a lab-on-a-chip, or a combination thereof. The bioanalyzer system for performing bioanalyzer measurements of cell culture samples can be automated. The bioanalyzer system can analyze a single sample by performing about 1 to about 20 determinations simultaneously. Exemplary non-limiting bioanalyzer measurements that can be performed by the bioanalyzer system include quantifying pH, CO2 partial pressure (pCO2), O2 partial pressure, glucose, lactate, glutamine, glutamate, ammonium, sodium, potassium, calcium, osmotic pressure, total cell density, viable cell density, cell viability, or a combination thereof. The bioanalyzer system can use a trypan blue exclusion test to determine viable cell density. Cell culture samples within the 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 determinations, or a combination thereof. DNA determinations can determine the size or quantity of DNA, or the size and quantity of DNA within a cell culture sample. RNA assays can quantify total RNA, mRNA, or both within a sample, determine the size of at least one small RNA within a sample, determine the purity and integrity of a sample, or a combination thereof. Protein assays can determine the size or quantity of at least one protein within a sample. Flow cytometry can be used to analyze protein expression, apoptosis, gene silencing, transfection monitoring, intracellular staining, extracellular staining, or a combination thereof.

[0347] In one aspect, cell culture nutrients can include, for example, glucose, glutamine, glutamate, sodium, potassium, calcium, O2, or a combination thereof.

[0348] In one aspect, cell culture metabolites can include, for example, acetate, acetate, lactate, lactate, ammonia, ethanol, lactate, CO2, or a combination thereof.

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

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

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

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

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

[0354] Fc glycans can play a role in immunogenicity, bioactivity, pharmacodynamics, and pharmacokinetics. Reusch and Tejada, Glycobiol. 25: 1325-34 (2015). A phenomenon that may occur is called non-glycosylated heavy chain (NGHC). NGHC variation can alter effector functions, such as opsonization. Opsonization involves the Fc portion 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 the Fc portion.

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

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

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

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

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

[0360] Table 6

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

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

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

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

[0365] In one embodiment, cells are cultured in large-scale containers or bioreactors where the levels of dissolved gases, including oxygen, are measured periodically. In one aspect, the period for measuring dissolved oxygen levels can be selected by the user. In another aspect, dissolved oxygen levels can be measured automatically over set time periods.

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

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

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

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

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

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

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

[0373] In another aspect, the main peak form of Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, produced by the cells can comprise between about 38% and about 65% of the total Fc-containing protein; the acidic variant of the Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, can comprise between about 20% and about 47% of the total Fc-containing protein; and the basic variant of the Fc-containing protein, such as an antibody capable of binding to IL-4 receptor, can comprise up to about 36% of the total Fc-containing protein. The percentage of Fc-containing protein with aglycosylated heavy chains can comprise between about 3% and 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 fractional value within these ranges.

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

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

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

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

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

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

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

[0381] Additional details based on FcR-containing proteins, such as antibodies capable of binding to the IL-4 receptor, are further described by Example 18 below, which illustrates various aspects of the invention but does not limit the invention in any way and can be applied to cells cultured under appropriate pCO2 conditions for controlling acidic species and unglycosylated heavy chain of dupilumab.

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

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

[0384] Centrifugation, depth and fine filtration

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

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

[0387] Table 7

[0388]

[0389]

[0390] g, standard gravity

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

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

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

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

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

[0396] Such centrifuge liquid can then be collected for further processing or filtered online through one or more depth filters to further clarify the sample. The most commonly used depth filters for bioprocessing are composed of diatomaceous earth, cellulose fibers and a positively charged resin binder. Unlike membrane filters, depth filters retain particles throughout the porous filter medium, allowing particles larger than and smaller than the pore size to be retained. It is believed that particle retention involves both size exclusion and adsorption via ionic, hydrophobic and other mechanisms. Filter fouling mechanisms can include pore blockage, filter cake formation and pore shrinkage. Non-limiting examples of depth filters that can be used in the context of the present disclosure include Millistak+X0HC, FOHC, DOHC, COHC, A1HC, B1HC depth filters (EMD Millipore), 3M TM Model 30 / 60ZA, 60 / 90ZA, VR05, VR07, degreasing depth filter (3M Corp.) 0.2 μm filter, such as Sartorius 0.45 / 0.2 μm Sartopore TM A double layer or Millipore SHR or SHC filter cartridge is typically placed after the depth filter. An Emphaze AEX Hybrid Purifier multi-mechanism filter can also be used to filter the depth filtered material. Other filters known to those skilled in the art can also be used.

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

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

[0399] Protein A chromatography

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

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

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

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

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

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

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

[0407]

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

[0409] Using an affinity wash buffer to reduce HCP content and / or HMW species can enable the use of fewer downstream chromatography steps to achieve the corresponding drug substance quality, for example, using a protein A column and an AEX column instead of a CEX column or HIC column; or using a protein A column followed by a mixed-mode column and an AEX column instead of a CEX column or HIC column. If the impurity content that can be achieved using another column is not matched by optimization of the affinity capture wash buffer alone, then additional specific gravity can be obtained by using a charged filter medium during the filtration step. This is usually performed after the viral inactivation step, during which several pH adjustments have been made and filtration for specific conditions is easy to incorporate into the downstream process. Filter media with multiple removal mechanisms are commercially available, including cationic, anionic, hydrophobic, and mixed-mode mechanisms. Because the charge density on the filter is generally lower when compared to packed bed chromatography, operating the filter under conditions where the product of interest does not bind increases the likelihood of impurity removal and high recovery.

[0410] Low pH maintenance

[0411] In some exemplary embodiments, a recovery process can also be the main points of reducing or deactivating the virus that may be present in the biological sample. During the recovery phase of production, any one or more of multiple viruses can be used to reduce / inactivation methods, including heat inactivation (pasteurization), pH inactivation, buffer / cleaning agent treatment, UV and gamma ray irradiation and adding some chemical inactivators, such as beta-propiolactone or for example as phenanthenoline copper described in U.S. Patent No. 4,534,972, all teaching contents of the patent are incorporated herein by reference. On the other hand, the virus interception filtration as a dedicated virus removal step can also be used and discussed below.

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

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

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

[0415] Anion exchange chromatography

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

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

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

[0419] In performing the separation, the initial protein composition (biological sample) can be contacted with the anion exchange material using any of a variety of techniques, such as batch techniques or chromatographic techniques.

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

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

[0422] In some embodiments, the amount of protein loaded onto the AEX resin (e.g., grams of protein per liter of resin) is between about 50 g / L and about 200 g / L, between about 100 g / L and about 150 g / L, less than about 120 g / L, about 50 g / L, about 55 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, about 80 g / L, about 85 g / L, about 90 g / L, about 95 g / L, about 100 g / L, about 150 g / L, about 120 g / L, about 15 ... g / L, about 170 g / L, about 175 g / L, about 180 g / L, about 185 g / L, about 190 g / L, about 195 g / L, or about 200 g / L.

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

[0424] In some embodiments, the sample can be further conditioned after neutralization and prior to contacting the AEX material. The sample, e.g., a virus inactivated pool, can be conditioned to between about 3.00 mS / cm and about 6.00 mS / cm, between about 3.00 mS / cm and about 4.00 mS / cm, between about 3.40 mS / cm and about 3.60 mS / cm, about 3.00 mS / cm, about 3.10 mS / cm, about 3.20 mS / cm, about 3.30 mS / cm, about 3.40 mS / cm, about 3.50 mS / cm, about 3.60 mS / cm, about 3.70 mS / cm, about 3.80 mS / cm, about 3.90 mS / cm, about 4.00 mS / cm, about 4. mS / cm, about 10 mS / cm, about 4.20 mS / cm, about 4.30 mS / cm, about 4.40 mS / cm, about 4.50 mS / cm, about 4.60 mS / cm, about 4.70 mS / cm, about 4.80 mS / cm, about 4.90 mS / cm, about 5.00 mS / cm, about 5.10 mS / cm, about 5.20 mS / cm, about 5.30 mS / cm, about 5.40 mS / cm, 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 can be conditioned using, for example, about 2 M sodium acetate, about 2 M Tris base, or a combination thereof.

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

[0426] In some embodiments, the AEX column is subjected to an equilibration step prior to contact with the sample and optionally after the pre-equilibration step. The equilibration step changes the pH and conductivity of the mobile phase to match the buffer excipients present in the loading material (sample). In some embodiments, the equilibration buffer may comprise about 50 mM Tris and about 60 mM acetate, having a pH between about 7.50 and about 7.70 and a conductivity between about 3.00 mS / cm and about 4.00 mS / cm. In some embodiments, the pH of the equilibration buffer is between about 7.40 and about 8.30, between about 7.50 and about 7.70, between about 7.55 and about 7.65, about 7.40, about 7.45, about 7.50, about 7.51, about 7.52, about 7.53, about 7.54, about 7.55, about 7.56, about 7.57, about 7.58, about 7.59, about about 7.60, about 7.61, about 7.62, about 7.63, about 7.64, about 7.65, about 7.66, about 7.67, about 7.68, about 7.69, about 7.70, about 7.75, about 7.80, about 7.85, about 7.90, about 7.95, about 8.00, about 8.05, about 8.10, about 8.15, about 8.20, about 8.25 or about 8.30. In some embodiments, the conductivity of the equilibration buffer is between about 3.00 mS / cm and about 6.00 mS / cm, between about 3.00 mS / cm and about 4.00 mS / cm, between about 3.40 mS / cm and about 3.60 mS / cm, about 3.00 mS / cm, about 3.10 mS / cm, about 3.20 mS / cm, about 3.30 mS / cm, about 3.40 mS / cm, about 3.50 mS / cm, about 3.60 mS / cm, about 3.70 mS / cm, about 3.80 mS / cm, about 3.90 mS / cm, about 4.00 mS / cm, about 4. .10 mS / cm, about 4.20 mS / cm, about 4.30 mS / cm, about 4.40 mS / cm, about 4.50 mS / cm, about 4.60 mS / cm, about 4.70 mS / cm, about 4.80 mS / cm, about 4.90 mS / cm, about 5.00 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 between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, about 100 cm / hr, about 110 cm / hr, about 120 cm / hr, about 130 cm / hr, about 140 cm / hr, about 150 cm / hr, about 160 cm / hr, about 170 cm / hr, about 180 cm / hr, about 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, or about 300 cm / hr. {In some exemplary embodiments, about three column volumes of equilibration buffer are used.}

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

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

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

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

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

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

[0433] The anionic 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.

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

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

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

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

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

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

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

[0441] Cation exchange chromatography

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

[0443] In performing cation exchange, a sample containing the protein of interest can be contacted with a cation exchange material using any of a variety of techniques (e.g., batch or chromatographic techniques), as described above for AEX. Cation exchange packed bed chromatography is based on ionic interactions between a binding entity (target protein or impurity) and functional groups immobilized on a chromatographic medium. Performance can vary depending on, 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 the content of HCCP and HMW product-related impurities, such as protein aggregates.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0457] Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S). Additional cationic materials include, but are not limited to: Capto SPImpRes, which are high-flow agarose beads; Capto S ImpAct; Grade F CM Hyper D, which are coated ceramic beads impregnated with a functionalized hydrogel (250 to 400 ionic groups μeq / mL); Eshmuno S, which is a hydrophilic polyvinyl ether basic matrix with an ionic capacity of 50 to 100 μM / mL; Nuvia C Prime, which is a hydrophobic cation exchange medium composed of a 55 to 75 με / mL macroporous, highly cross-linked hydrophilic polymer matrix; Nuvia S, which has a UNOspher basic matrix with 90 to 150 με / mL ionic groups; Poros HS, which are rigid polymeric beads with a backbone composed of cross-linked poly[styrene-divinylbenzene]; Poros XS, which are rigid polymeric beads with a backbone composed of cross-linked poly[styrene-divinylbenzene]; Toyo Pearl Giga Cap CM 650M, which are polymeric basic beads with an ionic capacity of 0.225 meq / mL; Toyo Pearl Giga Cap S650M, which are polymeric basic beads; Toyo Pearl MX TRP, which are polymeric basic beads; and EMD SE Hicap. It should be noted that CEX chromatography can be used with the MM resins described herein. In some embodiments, a CEX resin with a relatively high capacity is selected, for example, for loading up to about 100 g / L of resin. In some embodiments, a CEX resin with relatively high caustic stability is selected, for example, to prevent hydrolysis of cationic substrates.

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

[0459] Mixed-mode chromatography

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

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

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

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

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

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

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

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

[0468] In some embodiments, the wash buffer may comprise any of the pH values, NaCl, concentrations, or buffer salts of the equilibration buffers described above.

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

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

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

[0472] Hydrophobic interaction chromatography

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

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

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

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

[0477] As with ion exchange chromatography, HIC columns or HIC membrane devices can also be operated in elution mode, flow-through mode, or mixed mode, in which the product exhibits binding or interaction with the chromatography 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 with respect to AEX processing.) In some embodiments, the HIC step is performed in negative mode, in which process-related impurities are bound to an immobilized ligand and the protein of interest flows through.

[0478] Since hydrophobic interactions are strongest at high ionic strengths, this form of separation is preferably performed after a salt elution step such as those commonly used in conjunction with ion exchange chromatography. Alternatively, salt can be added to the sample before the HIC step is employed. High salt concentrations favor adsorption of proteins to the HIC column, but the actual concentration can vary widely depending on the nature of the protein of interest, the type of salt, and the specific HIC ligand selected. The various ions can be arranged in a so-called solvophobic series depending on whether they promote hydrophobic interactions (salting out) or disrupt water structure (chaotropic effects) and lead to a weakening of hydrophobic interactions. Cations are arranged in the order of Ba according to increasing salting out. 2+ , Ca 2+ Mg 2+ 、Li + 、Cs + 、Na + , K + , Rb + NH4 + , and the anion is discharged as PO4 according to the increasing chaotropic effect 3- 、SO4 2- 、CH3CO3 - 、CI - Br - 、NO3 - 、ClO4 - , I - 、SCN - .

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

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

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

[0482] In some embodiments, the concentration of protein loaded onto the HIC resin (e.g., grams of protein per liter of resin) is between about 60 g / L and about 180 g / L, between about 100 g / L and about 150 g / L, between about 180 g / L and about 200 g / L, less than about 180 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, about 80 g / L, about 85 g / L, about 90 g / L, about 95 g / L, about 100 g / L, about 15 ... g / L, about 170 g / L, about 175 g / L, about 175 g / L, about 180 g / L, about 185 g / L, about 190 g / L, about 195 g / L, or about 200 g / L.

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

[0484] In some embodiments, the HIC column is subjected to a pre-equilibration step prior to contact with the sample. The pre-equilibration step removes 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 between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, about 100 cm / hr, about 110 cm / hr, about 120 cm / hr, about 130 cm / hr, about 140 cm / hr, about 150 cm / hr, about 160 cm / hr, about 170 cm / hr, about 180 cm / hr, about 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, or about 300 cm / hr. In one exemplary embodiment, the linear velocity is about 200 cm / hr. In some exemplary embodiments, about three column volumes of pre-equilibration buffer are used.

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

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

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

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

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

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

[0491] Size exclusion chromatography

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

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

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

[0495] Virus retention filtration

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

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

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

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

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

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

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

[0503] Concentration and diafiltration

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

[0505] Ultrafiltration is described in detail in Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, NY, 1996); and in Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN 87762-456-9); the entire teachings of which are incorporated herein by reference. One filtration method is tangential flow filtration, which is described in the Millipore catalog entitled "Pharmaceutical Process Filtration Catalogue," pages 177-202 (Bedford, Mass., 1995 / 96), the entire teachings of which are incorporated herein by reference. Ultrafiltration is generally considered to mean filtration using filters with a pore size of less than 0.1 μm. By employing filters of such small pore size, the volume of the sample can be reduced by allowing the sample buffer to permeate the filter membrane pores, while proteins are retained on the membrane surface.

[0506] UF / DF typically involves three to four stages: initial concentration, diafiltration, secondary concentration, and final concentration. During initial concentration, the TMP forces water and salts through the membrane, reducing the liquid volume and thereby increasing protein concentration. The degree of concentration during initial concentration can be optimized to balance yield and protein stability.

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

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

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

[0510] The exemplary steps are again described in more detail. The first UF / DF step includes a WFI flush with a retentate volume of approximately 12 L / m 2 And the permeate is 40L / m 2 The feed flow rate is about 200 and 300 L / hr / m 2( The equilibration buffer was then stirred for 1 hr at 400 rpm for 2-3 hr. The equilibration buffer was stirred for 1 hr. The equilibration buffer was stirred for 2 hr. The equilibration buffer was stirred for 1 hr. The equilibration buffer was stirred for 2 hr. The equilibration buffer was stirred for 3 ... 2 , and the flow rate is between about 200 and 300LMH.

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

[0512] Next, an initial concentration step is performed using a load, such as a VRF pool. 2 The protein concentration after initial concentration can be between about 60 and 80 g / L. In an exemplary embodiment, the protein concentration in this step is about 70 g / L.

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

[0514] The volume of the diafiltration buffer used can be about or greater than 800V, the flow velocity is between about 200 and 350LMH, and the pressure is between about 10 and 30psi. In an exemplary embodiment, the flow velocity of the diafiltration is about 300LMH. In an exemplary embodiment, the pressure of the diafiltration is about 20psi. The protein concentration after the diafiltration can be between about 60 and about 80g / L. In an exemplary embodiment, the protein concentration after the diafiltration is about 70g / L.

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

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

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

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

[0519] Finally, after membrane polarization, there is a WFI flush step, applying 10 L / m 2 WFI.

[0520] preparation

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

[0522] If the filtered FCP protein concentration is above a specified target, it is diluted with FCP dilution buffer to adjust it to the target concentration. The FCP dilution buffer may comprise about 10 mM sodium acetate, about 25 mM arginine hydrochloride, and about 5% sucrose at a pH of about 5.3. In some embodiments, the concentration of sodium acetate in the FCP dilution buffer may be between about 2.0 mM and about 14.0 mM, between about 6.0 mM and about 12.0 mM, between about 8.0 mM and about 10.0 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6.0 mM, about 7.0 mM, about 8.5 mM, about 9.0 mM, about 10.0 mM, about 11.0 mM, about 12.0 mM, about 13.0 mM, about 14.0 mM, about 15.0 mM, about 16.0 mM, about 17.0 mM, about 18.0 mM, about 19.0 mM, about 20.0 mM, about 21.0 mM, about 22.0 mM, about 23.0 mM, about 24.0 mM, about 25.0 mM, about 26.0 mM, about 27.0 mM, about 28.0 mM, about 29.0 mM, about 30.0 mM, about 31.0 mM, about 32.0 mM, about 33.0 mM, about 34.0 mM, about 35.0 mM, about 36.0 mM, about 37.0 mM, about 38.0 mM, about In some embodiments, the concentration of arginine hydrochloride in the FCP dilution buffer can be between about 10 mM and about 40 mM, between about 15 mM and about 35 mM, between about 20 mM and about 30 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, or about 40 mM. In some embodiments, the concentration of sucrose in the FCP dilution buffer can be between about 1% and 10%, between about 2% and 9%, between about 3% and 8%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the pH of the FCP dilution buffer can be between about 4.0 and about 6.5, between about 4.5 and about 6.0, between about 5.0 and about 5.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, or about 7.0.

[0523] In some embodiments, FCP can be further combined with an excipient concentration buffer to achieve both a target dilution of the FCP protein and a target excipient concentration. The FCP excipient concentration buffer can comprise about 10 mM acetate, about 350 mM arginine hydrochloride, and about 70% (w / v) sucrose at a pH of about 5.3. In some embodiments, the concentration of acetate in the excipient concentration buffer can be between about 2.0 mM and about 14.0 mM, between about 6.0 mM and about 12.0 mM, between about 8.0 mM and about 10.0 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6.0 mM, about 7.0 mM, about 8.0 mM, about 9.0 mM, about 10.0 mM, about 12.0 mM, about 13.0 mM, about 14.0 mM, about 15.0 mM, about 16.0 mM, about 17.0 mM, about 18.0 mM, about 19.0 mM, about 20.0 mM, about 21.0 mM, about 22.0 mM, about 23.0 mM, about 24.0 mM, about 25.0 mM, about 26.0 mM, about 27.0 mM, about 28.0 mM, about 29.0 mM, about 30.0 mM, about 31.0 mM, about 32.0 mM, about 33.0 mM, about 34.0 mM, about 35.0 mM, about 36.0 mM, about 37.0 mM, about 38.0 m In some embodiments, the concentration of arginine hydrochloride in the excipient concentration buffer can be between about 200mM and about 500mM, between about 250mM and about 450mM, between about 300mM and about 400mM, about 200mM, about 250mM, about 300mM, about 350mM, about 400mM, about 450mM or about 500mM. In some embodiments, the concentration of sucrose in the excipient concentration buffer can be between 50% and 90%, between about 55% and 85%, between about 60% and 80%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, or about 85%. In some embodiments, the pH of the excipient concentration buffer can be between about 4.0 and about 6.5, between about 4.5 and about 6.0, between about 5.0 and about 5.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, or about 7.0.

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

[0525] The target concentration may be about 200.0 mg / mL. In some embodiments, the target concentration is between about 160.0 mg / mL and about 240.0 mg / mL, between about 180.0 mg / mL and about 220.0 mg / mL, between about 190.0 mg / mL and about 210.0 mg / mL, between about 195.0 mg / mL and about 205.0 mg / mL, between about 199.0 mg / mL and about 201.0 mg / mL, between about 199.5 mg / mL and about 200.5 mg / mL, between about 199.9 mg / mL and about 200.1 mg / mL, about 160.0 mg / mL, about 165.0 mg / mL, about 170.0 mg / mL, about 180.0 mg / mL, about 191.0 mg / mL, about 193.0 mg / mL, about 194.0 mg / mL, about 195.0 mg / mL, about 196.0 mg / mL, about 197.0 mg / mL, about 198.0 mg / mL, about 199.0 mg / mL, about 201.0 mg / mL, about 199.5 mg / mL and about 200.5 mg / mL, about 199.9 mg / mL and about 200.1 mg / mL, about about 175.0 mg / mL, about 180.0 mg / mL, about 185.0 mg / mL, about 190.0 mg / mL, about 195.0 mg / mL, about 199.0 mg / mL, about 199.5 mg / mL, about 199.9 mg / mL, about 200.0 mg / mL, about 200.1 mg / mL, about 200.5 mg / mL, about 201.0 mg / mL, about 205.0 mg / mL, about 210.0 mg / mL, about 215.0 mg / mL, about 220.0 mg / mL, about 225.0 mg / mL, about 230.0 mg / mL, about 235.0 mg / mL, or about 240.0 mg / mL.

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

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

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

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

[0530] Example

[0531] Example 1. Insulin supplementation

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

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

[0534] Table 9 (Insulin supplementation experimental conditions)

[0535]

[0536] Table 10 (Insulin Supplementation 2L Bioreactor Conditions)

[0537]

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

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

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

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

[0542] Example 2. Tyrosine Supplementation

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

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

[0545] Table 11

[0546]

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

[0548] Example 3. Phosphate Redistribution

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

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

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

[0552] Table 12

[0553]

[0554]

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

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

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

[0558] Example 4. Production bioreactor conditions

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

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

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

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

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

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

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

[0566]

[0567] Collection Overview

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

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

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

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

[0572] Collection preprocessing

[0573] In certain exemplary embodiments, the collection process may include an initial step that delays the enzymatic process and reduces aggregate formation. Initial recovery may be combined with the use of a pretreatment. Pretreatment is designed to improve the collection step by using additives that help remove cells and help reduce process-related impurity content, thereby reducing the burden on downstream chromatography steps. Pretreatment includes flocculation or precipitation, and both methods promote the association or agglomeration of smaller particles to form larger solids that can be more effectively removed. Non-limiting examples of these pretreatments include polymeric flocculants, chitosan, poly (ethyleneimine) and cationic salts such as calcium phosphate.

[0574] Examples of harvest pretreatments that can be used in the present invention are described more fully in the Examples below. In one aspect, this step can be performed by cooling the bioreactor to approximately (±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 fine filter binding during the process.

[0575] Example 5: Chitosan flocculant pretreatment

[0576] In one embodiment, chitosan is dissolved in 1% (w / v) acetic acid, forms 125mM stock solution of calcium phosphate by 500mM sodium phosphate and 800mM calcium chloride of equal portions simultaneously.The cell culture fluid that is used for polyethyleneimine (PEI) and chitosan derives from BRX161-10128, and the material that is used for calcium phosphate derives from two development bioreactors TD29-10437 and TD32-10438.Use acetic acid that the collection of cell culture f...

Claims

1. A method for producing dupilumab, comprising: (a) culturing cells expressing dupilumab using a cell culture medium comprising between about 0.09 and about 0.9 mM ornithine and / or between about 0.20 mM and about 0.9 mM putrescine, (b) harvesting the cells by centrifugation to separate cell debris from the clarified culture medium containing dupilumab; (c) subjecting the clarified medium to affinity chromatography; (d) subjecting the pooled dupilumab from the eluate of step (c) to viral inactivation at a pH of about 3 to about 4 and then adjusting the pH to about 5 to about 8; (e) subjecting the Dupilumab pooled from step (d) to anion exchange chromatography in flow-through mode; (f) subjecting the dupilumab pooled from the flow-through fractions of step (e) to cation exchange chromatography in a bind and elute mode; (g) subjecting the pooled dupilumab from the eluate of step (f) to hydrophobic interaction chromatography in flow-through mode; (h) subjecting the dupilumab pooled from the flow-through fractions of step (g) to virus-retaining filtration to produce dupilumab; and (i) collecting the dupilumab.

2. The method of claim 1, wherein the cell culture medium comprises one or more fatty acids.

3. The method of claim 2, wherein the one or more fatty acids are selected from the group consisting of linoleic acid, linolenic acid, lipoic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, capric acid, dodecanoic acid, caproic acid, lignoceric acid, myristic acid, caprylic acid, and combinations thereof.

4. The method of claim 1, wherein the culture medium comprises a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof.

5. The method of claim 1, wherein the culture medium comprises amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof.

6. The method of claim 1, further comprising the step of adding one or more point-of-use supplements to the cell culture medium.

7. A method for producing dupilumab, comprising the following steps: (a) culturing cells expressing dupilumab using a cell culture medium comprising between about 0.09 and about 0.9 mM ornithine and / or between about 0.20 mM and about 0.9 mM putrescine, (b) collecting the cells by centrifugation to separate cell debris from the clarified culture medium containing dupilumab; (c) subjecting the clarified medium to affinity chromatography; (d) subjecting the pooled dupilumab from the eluate of step (c) to viral inactivation at a pH of about 3 to about 4 and then adjusting the pH to about 5 to about 8; (e) subjecting the pooled dupilumab from step (d) to cation exchange chromatography in bind and elute mode; (f) subjecting the pooled dupilumab from the eluate of step (e) to anion exchange chromatography in flow-through mode; and (g) subjecting the dupilumab pooled from the flow-through fractions of step (f) to virus-retaining filtration to produce dupilumab.

8. The method of claim 7, wherein the cell culture medium comprises one or more fatty acids selected from the group consisting of linoleic acid, linolenic acid, lipoic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, capric acid, dodecanoic acid, caproic acid, lignoceric acid, myristic acid, caprylic acid, and combinations thereof.

9. The method of claim 7, wherein the culture medium comprises a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof.

10. The method of claim 7, wherein the culture medium comprises insulin.

11. The method of claim 7, wherein the culture medium comprises amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof.

12. The method of claim 7, further comprising the step of adding one or more point-of-use supplements to the cell culture medium.

13. The method of claim 12, wherein the point-of-use additive comprises one or more of the following: NaHCO3, Na2HPO4, taurine, glutamine, poloxamer 188, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4 EDTA, and trisodium citrate EDTA.

14. The method of claim 7, wherein the culture medium does not contain hydrolysate.

15. A method for producing dupilumab, comprising the following steps: (a) culturing cells expressing dupilumab in a large-scale bioreactor, wherein the bioreactor comprises one or more optical probes for measuring dissolved gases; (b) culturing the cells in a culture medium comprising between about 0.09 and about 0.9 mM ornithine and / or between about 0.20 mM and about 0.9 mM putrescine; and (c) Production of dupilumab.

16. The method of claim 15, wherein the optical probe is used to measure dissolved oxygen.

17. The method of claim 16, further comprising the step of agitating the culture medium with one or more impeller assemblies, wherein an uppermost impeller is positioned below the surface of the initial working volume of the bioreactor.

18. The method of claim 16, further comprising the step of adjusting the dissolved oxygen level by sparging the culture medium.

19. The method of claim 15, further comprising adjusting the pCO2 content by sparging the culture medium.

20. The method according to claim 15, further comprising the step of adding taurine or hypotaurine to the culture medium.

21. The method of claim 20, further comprising the step of adding at least one recombinant growth factor.

22. The method of claim 21, further comprising the step of adding one or more of the following: adenosine, guanosine, cytidine, uridine, thymidine and hypoxanthine.

23. The method of claim 22, further comprising the step of adding a fatty acid comprising one or more of the following: linoleic acid, linolenic acid, lipoic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, capric acid, dodecanoic acid, caproic acid, lignoceric acid, myristic acid, and caprylic acid.

24. The method of claim 23, further comprising the step of adding one or more salts selected from the group consisting of divalent cations such as calcium ions, magnesium ions, and combinations thereof.

25. The method of claim 24, further comprising the step of adding an amino acid having a non-polar side chain.

26. The method according to claim 25, further comprising the step of adding a basic amino acid.

27. The method of claim 26, further comprising the step of adding nucleosides, salts of divalent cations, tocopherols and vitamins.

28. A method for producing dupilumab in a modified bioreactor, comprising the following steps: (a) culturing cells expressing dupilumab in a container or bioreactor, wherein the bioreactor comprises at least one inline capacitance probe; (b) culturing the cells in a medium comprising one or more polyamines; and (c) Production of dupilumab.

29. The method according to claim 28, further comprising the steps of: i) applying an electric field to said cells cultured in a bioreactor; ii) measuring capacitance; and iii) Correlate capacitance with viable cell density.

30. The method of claim 29, further comprising the step of transferring the cells when the final VCD reaches a target cell density.

31. The method of claim 30, further comprising adjusting the initial VCD of the seed expansion to at least 2.5×10 5 cells / mL step.

32. A method comprising: (a) culturing cells expressing an anti-IL-4Rα antibody or an antigen-binding fragment thereof using a cell culture medium comprising between about 0.09 and about 0.9 mM ornithine and / or between about 0.20 mM and about 0.9 mM putrescine, (b) collecting the cells by centrifugation to separate cell debris from the clarified culture medium containing the anti-IL-4Rα antibody or antigen-binding fragment thereof; (c) subjecting the clarified medium 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 and subsequently adjusting the pH to about 5 to about 8; (e) subjecting the anti-IL-4Rα antibodies or antigen-binding fragments thereof pooled from step (d) to anion exchange chromatography in flow-through mode; (f) subjecting the anti-IL-4Rα antibodies or antigen-binding fragments thereof pooled from the flow-through fractions of step (e) to cation exchange chromatography in a bind and elute mode; (g) subjecting the anti-IL-4Rα antibodies or antigen-binding fragments thereof pooled from the eluate of step (f) to hydrophobic interaction chromatography in flow-through mode; (h) subjecting the anti-IL-4Rα antibody or antigen-binding fragment thereof pooled from the flow-through fraction of step (g) to virus-retaining filtration to produce the anti-IL4Rα antibody or antigen-binding fragment thereof; and (i) collecting the anti-IL-4Rα antibody or the antigen-binding fragment thereof.

33. The method of claim 32, wherein the cell culture medium comprises one or more fatty acids.

34. The method of claim 33, wherein the one or more fatty acids are selected from the group consisting of linoleic acid, linolenic acid, lipoic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, capric acid, dodecanoic acid, caproic acid, lignoceric acid, myristic acid, caprylic acid, and combinations thereof.

35. The method of claim 32, wherein the culture medium comprises a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof.

36. The method of claim 32, wherein the culture medium comprises amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof.

37. The method of claim 32, further comprising the step of adding one or more point-of-use supplements to the cell culture medium.

38. The method of claim 37, wherein the point-of-use additive comprises one or more of the following: NaHCO3, Na2HPO4, taurine, glutamine, poloxamer 188, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4 EDTA, and trisodium citrate EDTA.

39. The method of claim 32, wherein the culture medium is free of hydrolysates.

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

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

42. A method for treating a disease or condition associated with IL-4R activity, comprising administering to a patient dupilumab produced according to the method of any one of claims 1 to 39.

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

Images