Manufacturing process of high-titer antibody

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 of related applications

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

[0003] sequence list

[0004] This application contains a sequence list, which has been submitted electronically in .xml format and is hereby incorporated by full reference. A copy of the .xml file created on January 31, 2023, is named sequencelist.xml and is 180,965 bytes in size. Technical Field

[0005] This application relates to the production of recombinant proteins in high-productivity cells using serum-free culture media for large-scale production, and to improved systems and methods for measuring and maintaining cell culture conditions, collecting and purifying proteins with reduced heterogeneity and improved quality and higher yield. Background Technology

[0006] The manufacturing process for 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 modify protein production conditions to optimize the product's titer, batch size, yield, or quality. In turn, increased and optimized titers, batch sizes, and yields require optimization of collection and purification processes to handle the increased titers and loadings while maintaining acceptable quality properties.

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

[0008] Furthermore, measuring and maintaining optimized cell culture conditions is critical and requires specially designed equipment, including probes and sensors for measuring dissolved gases such as oxygen. However, the use of large-scale vessels and bioreactors can impact the equipment used to measure cell culture conditions, including its accuracy and maintenance requirements. Due to the stringent regulations of the biopharmaceutical industry, irregularities in measurements may require investigation, potentially necessitating 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 offer improved accuracy and reduced maintenance requirements.

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

[0010] To handle higher titers of drug products, improved methods and systems have been developed for the manufacture, collection, and purification of high-titer antibody drug products. For example, increased drug product titers and drug loading can exceed the capacity of certain chromatographic purification processes and alter viscosity, adversely affecting the transfer of the drug product along different chromatographic steps. Therefore, there is a need for improved methods that address not only individual purification steps but also the entire manufacturing process. Summary of the Invention

[0011] This disclosure provides an improved method for large-scale production of anti-IL4Rα antibodies in high-productivity cells. Improved cell culture media and methods have been developed to maintain optimal cell culture conditions to increase titers and improve quality. Improved methods have also been developed for collecting and purifying the increased protein loading while increasing overall yield, reducing manufacturing and operational complexity, and improving the overall quality of the active pharmaceutical ingredient and the formulated pharmaceutical 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 complementarity-determining region (HCDR) sequences comprising SEQ ID NO:3, 4, and 5 and three light chain complementarity-determining region (LCDR) sequences comprising SEQ ID NO:6, 7, and 8.

[0013] In some embodiments, the protein of interest is an antigen-binding protein, a blocking antibody, or a receptor antagonist (e.g., interleukin-4 receptor α (IL-4Rα)). In some aspects, the protein of interest is a protein having an Fc domain. Therefore, 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), a ScFv molecule, etc.

[0014] In some embodiments, the manufacturing process includes culturing cells capable of expressing anti-IL4Rα antibodies or receptor antagonists as described above in a cell culture medium containing a chemically defined basal medium (such as a custom-prepared or commercially available basal medium). In another embodiment, the complete culture medium is serum-free (“serum-free” medium) and / or free of hydrolysis products. In yet another embodiment, the method includes the step of adding one or more of the use-point additives described below to the cell culture medium. In some aspects, the use-point additives described below may also be included in the cell culture medium at the outset.

[0015] It has been found that polyamine-supplemented media (“PS media”) increase cell viability and density, reduce cell doubling time, and allow cells grown in such supplemented media to produce high-titer proteins. PS media, with or without serum and / or supplemental hydrolysates, have been found to provide restored cell viability and density, cell doubling time, and high-titer protein yields.

[0016] In one embodiment, cells cultured in PS medium have an average doubling time of no more than 30 hours. In another aspect, the cell doubling time is no more than 24 hours. Similarly, including polyamines in serum-free medium allows cultured cells to achieve a higher viable cell density (VCD) compared to excluding polyamines.

[0017] In one embodiment, the cell culture medium, which is free of serum and / or hydrolysis products, contains one or more polyamines selected from the group consisting of ornithine, putrescine, spermine, spermidine, or combinations thereof.

[0018] In one embodiment, the PS medium is serum-free and contains ornithine at a concentration between 30 μM and 900 μM. In one aspect, the ornithine is present in 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. The concentrations (expressed in micromoles per liter) of the PS medium are 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. Ornithine may be present in the medium at concentrations from about 0.09 mM to about 0.9 mM. In some embodiments, the PS medium contains ≤7.5 g / L of hydrolysis products. In some embodiments, the PS medium does not contain any hydrolysis products.

[0019] In one embodiment, the PS culture medium is serum-free and contains putrescine at a concentration between 30 μM and 900 μM. In one aspect, the putrescine is present in concentrations 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 μM. The medium is present in concentrations of 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 (expressed in micromoles per liter). Putrescine may be present in concentrations of about 0.20 mM to about 0.714 mM. In one embodiment, the PS 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 medium contains ≤7.5 g / L of hydrolysis products. In some embodiments, the PS medium does not contain any hydrolysis products.

[0020] In one embodiment, the PS medium is serum-free and contains spermine at a concentration between 10 μM and 900 μM. In one aspect, the spermine is present in concentrations 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, and 588. The PS medium is present in concentrations (expressed in micromoles per liter) of 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. In some embodiments, the PS medium contains ≤7.5 g / L of hydrolysis products. In some embodiments, the PS medium does not contain any hydrolysis products.

[0021] In one embodiment, the PS culture medium is serum-free and contains spermidine at a concentration between 10 μM and 900 μM. In one aspect, the spermidine is present in concentrations 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 μM. The medium is present at concentrations (expressed in micromoles per liter) of 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. 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 hydrolysis products. In some embodiments, the medium contains ≤16 g / L of hydrolysis products. In some embodiments, the PS medium contains no hydrolysis products.

[0022] In one embodiment, the culture medium comprises a mixture of amino acids selected from the group consisting of (notably, glutamine is an exception, which may be added back to the culture medium as a point-of-use additive): 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.

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

[0024] In some embodiments, the culture medium contains one or more fatty acids. In one aspect, the culture medium contains a mixture of a fatty acid (or a fatty acid derivative) and α-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, hexanoic acid, teicosanoic acid, myristic acid, and caprylic acid, and may also include lipoic acid derived from the fatty acid.

[0025] In some embodiments, the culture medium contains one or more nucleosides. In one aspect, the nucleosides are 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 consisting 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 salts of phosphate.

[0027] In some embodiments, the cell culture medium comprises any one or more of the following: NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4 EDTA, trisodium citrate, and combinations thereof. In one aspect, the method comprises the step of adding one or more of the following point-of-use chemicals selected from the group consisting of: NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4 EDTA, trisodium citrate, and combinations thereof to the cell culture medium.

[0028] In some embodiments, the additive used comprises taurine, phosphate esters, poloxamer 188, and combinations thereof. In some aspects, the additive used may be included in the culture medium at the beginning.

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

[0030] In some embodiments, this disclosure provides a method for generating an anti-IL-4Rα antibody or an antigen-binding fragment thereof by employing 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 described in this disclosure; and (4) expressing the protein of interest in the cells, wherein the protein of interest is secreted into the culture medium. The biotherapeutic protein may be a blocking antibody or receptor antagonist (e.g., blocking IL-4Rα and IL-13) and / or an antigen-binding protein, which may contain 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 cells are mammalian cells, avian cells, insect cells, yeast cells, or bacterial cells. In one aspect, the cells are mammalian cells suitable for producing recombinant proteins, such as CHO cells or the derivative CHO-K1. In some aspects, the cells express proteins of interest, such as biotherapeutic proteins. In some aspects, the cells used to produce the proteins are mammalian cells capable of producing biotherapeutic agents, such as CHO, HEK293, and BHK cells, or any derivatives thereof. In one embodiment, the cells are CHO cells, such as CHO-K1 cells.

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

[0034] In some embodiments, the cell culture medium is: (1) 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 additionally 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; and (5) contains at least about 40 mM or at least about 70 mM 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; (6) a mixture containing tocopherol and optionally fatty acids; (7) a mixture containing nucleosides, including adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; and (8) a mixture containing calcium salts, magnesium salts, phosphates, and combinations thereof.

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

[0036] In some embodiments, this disclosure provides a method for culturing cells in a serum-free culture 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; and (3) a mixture of at least about 40 mM or at least about 70 mM amino acids, including alanine, arginine, asparagine, aspartic acid, cysteine, and glutamine. The mixture comprises (3) amines, 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 one-third of the doubling time of cells cultured in a similar cell culture medium containing less than 0.09 ± 0.015 mM polyamines. In another embodiment, the cell culture is able to achieve a viable cell density at least 3 times higher than that of similar cell cultures in similar cell culture media 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 contains ≤7.5 g / L of hydrolysis products; and in another embodiment, the culture medium does not contain hydrolysis products.

[0037] In some embodiments, the protein of interest is generated by the following steps: (1) introducing a nucleic acid sequence encoding the protein of interest (such as an antibody or other antigen-binding protein) into CHO cells; (2) selecting cells carrying the nucleic acid sequence; and (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; and (c) a mixture of at least 40 mM or at least 70 mM amino acids. The medium includes one or more of the following: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (d) a mixture of tocopherols and optionally fatty acids; (e) a mixture of nucleosides, including one or more of the following: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; and (f) a salt, including one or more of the following: calcium salts, magnesium salts, phosphates, and combinations thereof; and (4) expression of the protein of interest in CHO cells, wherein the protein of interest is secreted in a culture medium. In some embodiments, the serum-free cell culture medium may include ≤7.5 g / L of hydrolysate; or in other embodiments, it may not include hydrolysate at all.

[0038] In some embodiments, the protein of interest is generated by the following steps: (1) introducing a nucleic acid sequence encoding the protein of interest (such as an antibody or other antigen-binding protein) into CHO cells; (2) selecting cells carrying the nucleic acid sequence; and (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; and (c) a mixture of at least 40 mM or at least 70 mM amino acids. The mixture comprises one or more of the following: 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; (d) a mixture of tocopherols and fatty acids; (e) a mixture of nucleosides, comprising one or more of the following: adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations thereof; and (f) a salt, comprising one or more of the following: calcium salts, magnesium salts, and phosphates; and (4) expression of the protein of interest in CHO cells, wherein the protein of interest is secreted in a culture medium. In some embodiments, the serum-free cell culture medium may include ≤7.5 g / L of hydrolysate; or in other embodiments, it may not include hydrolysate at all.

[0039] This disclosure provides an improved seed-raising method for increasing titer. In one embodiment, with a titer of 1.7 × 10⁻⁶... 5 Up to 2.3×10 5 Compared to the standard initial viable cell density (VCD) in the range of cells / mL, the initial VCD at each step (N-5 to N-1) increased to approximately 3.5 × 10⁻⁶ cells / mL. 5 Approximately 5.43 × 10 5Cells / mL. In some aspects, the initial VCD (in N-5 to N-1) was approximately 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 the surrogate initial VCD in standard seed expansion cultures. In one aspect, optimized seed expansion cultures resulted in a 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 1%, 17%, 18%, 19%, or 20% increase in final titer (g / L). In one aspect, the peak lactate observed in the final production vessel was not substantially different from that in standard seed expansion cultures.

[0040] This disclosure provides 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 in the biopharmaceutical industry.

[0041] This disclosure provides improved methods and systems for measuring air bubbling, agitation, and dissolved gases. More specifically, this disclosure provides optimized dissolved gas concentrations at various time intervals, and improved bioreactors with optimized agitation rates and operating volumes to provide necessary agitation while minimizing shear stress.

[0042] In some exemplary embodiments, the initial volume, air bubbling, and stirring setpoints in containers and bioreactors including various sensors are optimized during the growth and production phases of seed-scaled culture to improve process stability by minimizing shear stress on the cell culture and optimizing live cell density (VCD), dissolved oxygen, carbon dioxide, and pH at various time points.

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

[0044] This 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. Electrochemical probes are more commonly used in containers and bioreactors than optical probes because they have long been proven to have a wider linear range and faster reaction times compared to optical probes or sensors. However, electrochemical dissolved oxygen probes require extensive maintenance due to frequent membrane and electrolyte solution changes and require long polarization times. Furthermore, electrochemical probes are known to provide noisy readings due to bubble interference, as evidenced in the large-scale bioreactors provided in the examples below. This noise can cause deviations beyond the normal operating range and is attributed to stringent industry regulations, requiring investigation and verification through the quality assurance systems of biopharmaceutical companies. Studies should be conducted on transient high-side deviations typically attributed to bubbling interference to limit the source of manufacture.

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

[0046] Therefore, this disclosure provides an improved system and method for reducing signal noise in electrochemical probes, which reduces or eliminates false alarms attributable to bubble interference. Additionally, this disclosure provides a system and method for improving the accuracy of optical probes used to measure dissolved gases in large-scale containers and bioreactors, requiring less maintenance and being less susceptible to bubble interference compared to conventional electrochemical probes. As discussed in more detail below, improved data processing methods are used to develop containers and bioreactors with both electrochemical and optical probes to optimize the accuracy of measurements while minimizing maintenance by reducing or eliminating unwanted offset events outside the normal operating range, which requires lengthy quality studies in the highly regulated biopharmaceutical industry.

[0047] In one embodiment, this disclosure provides a method for measuring the VCD of cells cultured in a bioreactor by the following steps: applying an electric field to the cells cultured in the bioreactor; measuring the capacitance; and establishing a correlation between the measured capacitance and the live cell density.

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

[0049] To collect and purify the increased protein products, new and improved methods are developed that not only accommodate the increased protein yield but also improve the overall yield percentage. These methods include, for example, optimized collection pretreatment, protein A chromatography, virus inactivation, anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, virus-retaining filtration, concentration and percolation, and drug substance conditioning, which are further described below.

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

[0051] In one aspect, the antibody from step (h) is further concentrated and percolated using a percolation buffer with a pH between 4.0 and 4.5.

[0052] In one aspect, the percolation buffer contains approximately 4 mM acetate to approximately 6 mM acetate.

[0053] In one aspect, the collection pretreatment includes subjecting the antibody to an 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, affinity chromatography is protein A chromatography.

[0055] In one aspect, the collection in step (b) includes centrifugation and deep filtration.

[0056] In one aspect, antibodies are collected in a 3kL to 25kL bioreactor.

[0057] In one aspect, the loading pH of the protein A column is between 7 and 8. In another aspect, the loading pH of the protein A column is between 6 and 8. In yet another aspect, the loading pH of the protein A column is approximately 6.

[0058] In an alternative exemplary embodiment, the method includes: (a) culturing cells expressing an anti-IL-4Rα antibody or an antigen-binding fragment thereof; (b) subjecting the cells to an 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 medium containing the anti-IL-4Rα antibody or an antigen-binding fragment thereof; (d) subjecting the clarified medium to affinity chromatography; and (e) subjecting the anti-IL-4Rα antibody or an antigen-binding fragment thereof collected from the eluent of step (d) to viral inactivation at a pH of about 3 to about 4.5 and subsequently adjusting the pH to... (e) passing the anti-IL-4Rα antibody or its antigen-binding fragment collected from step (e) through anion exchange chromatography in flow-through mode; (g) passing the anti-IL-4Rα antibody or its antigen-binding fragment collected from the flow-through fraction of step (f) through cation exchange chromatography in binding and elution mode; (h) passing the anti-IL-4Rα antibody or its antigen-binding fragment collected from the eluent of step (g) through hydrophobic interaction chromatography in flow-through mode; and (i) passing the anti-IL-4Rα antibody or its antigen-binding fragment collected from the flow-through fraction of step (h) through virus-retaining filtration, thereby producing the anti-IL-4Rα antibody or its antigen-binding fragment.

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

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

[0061] In one aspect, the anti-IL-4Rα antibody or its antigen-binding fragment comprises three heavy chain complementarity-determining region (HCDR) sequences comprising SEQ ID NO:3, 4 and 5 and three light chain complementarity-determining region (LCDR) sequences comprising SEQ ID NO:6, 7 and 8.

[0062] In one aspect, the anti-IL-4Rα antibody or its antigen-binding fragment comprises a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) containing 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 this disclosure will be better understood and appreciated when considered in conjunction with the following description and accompanying drawings. While the following description indicates various embodiments and several specific details thereof, it is given 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 invention, and additional enumerated embodiments describing further aspects of this disclosure are provided below. Attached Figure Description

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

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

[0067] Figure 3 This indicates the effect of insulin supplementation according to an exemplary embodiment on the survival rate of cells expressing dupilumab.

[0068] Figure 4 The effect of insulin supplementation according to an exemplary embodiment on the reduction of ammonia content in cultures containing cells expressing dupilumab is illustrated.

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

[0070] Figure 6 The effects of insulin supplementation with a analyzer on high molecular weight species on day 13 are illustrated according to an exemplary embodiment.

[0071] Figure 7 The effect of insulin supplementation with a analyzer on the level of non-reducing purity on day 13 is shown according to an exemplary embodiment.

[0072] Figure 8 The effect of insulin supplementation arrangements using a analyzer on non-glycosylated heavy chain (NGHC) content is shown according to an exemplary embodiment.

[0073] Figure 9 The effect of tyrosine supplementation according to an exemplary embodiment on titers and ammonia content in cell cultures expressing plinomab is shown.

[0074] Figure 10 The effects of phosphate redistribution strategies 6 and 8 according to exemplary embodiments on dupilumab titers (g / L) are shown.

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

[0076] Figure 12A A contour plot showing a central synthetic design (CCD) model for predicting host cell protein clearance factor (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 of a CCD model for predicting host cell protein clearance factors according to an exemplary embodiment is shown.

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

[0080] Figure 14 The diagram illustrates the initial and final viable cell density (VCD) of an average standard seed expansion culture and an optimized seed expansion culture with an increased initial VCD compared to a standard seed expansion culture, according to an exemplary embodiment.

[0081] Figure 15 This illustrates a reduction in biomass in a 10kL production bioreactor according to an exemplary embodiment, the production bioreactor being used to culture cells using an optimized seed expansion culture with an increased initial VCD compared to a standard seed expansion culture.

[0082] Figure 16A and Figure 16B The final titer (g / L) of the 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 is shown.

[0083] Figure 17 The illustration shows a bivariate linear regression of cell line A (CLA) according to an exemplary embodiment of the present invention, with respect to in-line capacitance measurements and offline VCD measurements during seed expansion culture.

[0084] Figure 18 The diagram illustrates a bivariate linear regression of the CLA (capacitance measurement) during the N-3, N-2, and N-1 seed expansion culture stages according to an exemplary embodiment of the present invention, along with inline capacitance measurements and offline VCD measurements.

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

[0086] Figure 20 The illustration shows a bivariate linear regression of cell line B (CLB) according to an exemplary embodiment of the present invention, with respect to in-line capacitance measurements and offline VCD measurements during seed expansion culture.

[0087] Figure 21 The diagram illustrates a bivariate linear regression of CLB during the N-3, N-2, and N-1 seed expansion culture stages according to an exemplary embodiment of the present invention, along with inline capacitance measurements and offline VCD measurements.

[0088] Figure 22 The diagram shows predicted values ​​of CLB during the N-3, N-2, and N-1 seed expansion culture stages according to an exemplary embodiment of the invention, and along-line VCD measurements, determined using the along-line capacitance measurements and correlation equations presented in Figure 24. Dots (circles) indicate VCD values ​​measured offline using a bioanalyzer.

[0089] Figure 23 The diagram illustrates a bivariate linear regression of CLA and CLB during seed expansion culture and along with inline capacitance and offline VCD measurements, according to an exemplary embodiment of the present invention.

[0090] Figure 24A , Figure 24B and Figure 24C The diagram shows predicted values ​​of CLA and inline VCD measurements during the N-3, N-2, and N-1 seed expansion culture stages according to an exemplary embodiment of the present invention, using... Figure 17 The measured values ​​of the along-line capacitance presented in the figure and Figure 17 ( Figure 24A ), Figure 20 ( Figure 24B )and Figure 23 ( Figure 24C The correlation equation presented in the diagram is used to determine the VCD value. The dots (circles) indicate VCD values ​​measured offline using a bioanalyzer.

[0091] Figure 25A , Figure 25B and Figure 25C The diagram shows predicted values ​​(black traces) of CLB during the N-3, N-2, and N-1 seed expansion culture stages according to an exemplary embodiment of the present invention, measured using inline VCD. Figure 21 The measured values ​​of the along-line capacitance presented in the figure and Figure 20 ( Figure 25A ), Figure 17 ( Figure 25B )and Figure 23 ( Figure 25C The correlation equation presented in the diagram is used to determine the VCD value. The dots (circles) indicate VCD values ​​measured offline using a bioanalyzer.

[0092] Figure 26A and Figure 26B This diagram illustrates predicted values ​​of CLB during the seed expansion culture and production stages according to an exemplary embodiment of the present invention, using in-line VCD measurements. Figure 20 The in-line capacitance measurements and correlation equations presented in the figures determine the in-line capacitance. Figures A and B show data from two independent cultures, each measured using a single probe. Figure 26A In the diagram, the line plots the predicted values ​​of the VCD measurements along the line, and the dots (circles) indicate the VCD values ​​measured offline using a bioanalyzer. Figure 26B In the diagram, the line plots the predicted value of the VCD measurement along the line, and the dots (circles) indicate the VCD value measured offline using a bioanalyzer.

[0093] Figure 27A The host cell protein values ​​and dupilumab yield are shown after acid precipitation of dupilumab cell culture medium with 1M phosphate according to an exemplary embodiment.

[0094] Figure 27B The host cell protein values ​​and dupilumab yield are shown after acid precipitation of dupilumab cell culture medium with 1.75M acetic acid according to an exemplary embodiment.

[0095] Figure 28 A schematic diagram illustrating an exemplary overview of the use of depth filters, fine filters, and protective filters according to an exemplary embodiment.

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

[0097] Figure 30 A schematic diagram illustrating an exemplary purification step of an optimized process according to an exemplary embodiment is shown.

[0098] Figure 31 A schematic diagram illustrating an exemplary purification step of an optimized process according to an exemplary embodiment is shown.

[0099] Figure 32 The observations are shown from a model generated by a multivariate study of risk factors and responses to affinity capture and viral inactivation according to an exemplary implementation.

[0100] Figure 33The HCP content in affinity capture aggregates is shown according to an exemplary embodiment when using various affinity wash buffers.

[0101] Figure 34 The HCP content is shown after AEX or HIC treatment downstream of affinity capture chromatography using various affinity wash buffers, according to an exemplary embodiment.

[0102] Figure 35 The diagram illustrates the protein yield and HMW species in affinity capture aggregates when using various affinity wash buffers according to exemplary embodiments.

[0103] Figure 36 The observations are shown from a model generated by a multivariate study of AEX risk factors and responses according to an exemplary implementation.

[0104] Figure 37 The observations are shown from a model generated by a multivariate study of CEX risk factors and responses according to an exemplary implementation.

[0105] Figure 38 The observations are shown from a model generated by a multivariate study of HIC risk factors and responses according to an exemplary implementation.

[0106] Figure 39 The observations are shown from a model generated by a multivariate study of VRF risk factors and responses according to an exemplary implementation.

[0107] Figure 40 The observations are shown from a model generated by a multivariate study of UF / DF risk factors and responses according to an exemplary implementation.

[0108] Figure 41 The titer of a 500L confirmatory batch of dupilumab according to an exemplary embodiment is shown compared to a 10,000L process performance validation batch and a 2L small-scale bioreactor production batch.

[0109] Figure 42 The step yield of a unit operation is shown according to an exemplary embodiment of a 500L validation batch compared to a 10,000L process performance verification batch.

[0110] Figure 43 The HMW content of a unit operation is shown for a 500L validation batch compared to a 10,000L process performance verification batch according to an exemplary embodiment.

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

[0112] Figure 45 An embodiment of a step-by-time stirring and air bubbling rate according to an exemplary implementation is shown.

[0113] Figure 46 This illustration shows an example of applying the Agile filter to filter signals from cell cultures at a sampling rate of 59 seconds and a sample window of approximately 10 minutes, according to an exemplary embodiment. Label "A" indicates low lateral offset for smoothed signal retention. Label "B" indicates good consistency between the smoothed and unfiltered signals for anticipated process perturbations.

[0114] Figure 47 This illustration shows an example of applying the Agile filter to filter signals from cell cultures at a sampling rate of 59 seconds and a sample window of approximately 33 minutes, according to an exemplary embodiment. Label "A" indicates low lateral offset for smoothed signal removal. Label "B" indicates good consistency between the smoothed signal and the unfiltered signal for anticipated process perturbations.

[0115] Figure 48 This illustration shows an example of applying a Savitzky-Golay filter to filter signals from cell cultures at a sampling rate of 59 seconds and a sample window of approximately 33 minutes, according to an exemplary embodiment. Label "A" indicates noise retention relative to the filtered signal. Label "B" indicates good consistency between the smoothed signal and the unfiltered signal for the intended process perturbation.

[0116] Figure 49 The following is illustrated according to an exemplary embodiment: Figures 46 to 48 The superposition and comparison of filtered signals.

[0117] Figure 50 The first-round dissolved oxygen trend is shown according to an exemplary embodiment (where the light-colored line represents electrochemical, the medium-colored line represents optical probe B, the darkest line represents optical probe A, and the dashed lines represent the set point and the upper and lower limits of the normal operating range).

[0118] Figure 51 The second round of dissolved oxygen trends according to an exemplary embodiment is shown (where the light-colored line represents electrochemical, the medium-colored line represents optical probe B, the darkest line represents optical probe A, and the dashed lines represent the set point and the upper and lower limits of the normal operating range).

[0119] Figure 52 The third-round dissolved oxygen trend is shown according to an exemplary embodiment (where the light-colored line represents electrochemical, the medium-colored line represents optical probe B, the darkest line represents optical probe A, and the dashed lines represent the set point and the upper and lower limits of the normal operating range).

[0120] Figure 53The fourth round of dissolved oxygen trends according to an exemplary embodiment is shown (where the light-colored line represents electrochemical, the medium-colored line represents optical probe B, the darkest line represents optical probe A, and the dashed lines represent the set point and the upper and lower limits of the normal operating range).

[0121] Figure 54 Dissolved oxygen data are shown for the first to third rounds using an optical probe B with an anti-bubble optical cap, and for the fourth round using a standard optical cap, according to an exemplary embodiment.

[0122] Figure 55 The diagram shows dissolved oxygen data measured during the 'start,' 'middle,' and 'end' time periods of the second round of detection, as part of the offset assessment, according to an exemplary embodiment (where the light-colored line represents electrochemical, the medium-colored line represents optical probe B, the darkest line represents optical probe A, and the bottom line represents electrochemical with an Agile filter (59s sampling rate and preset sample window)).

[0123] Figure 56 The diagram illustrates the first round of probe B offset correction according to an exemplary embodiment (where the medium-colored line represents optical probe B, the darkest line represents optical probe B adjusted upwards by 3.0%, the light-colored line represents electrochemical probe B with an Agile filter (59s sampling rate and preset sample window), and the dashed lines represent the set point and upper / lower limits of the normal operating range).

[0124] Figure 57 The diagram illustrates the third round of probe B offset correction according to an exemplary embodiment (where the medium-colored line represents optical probe B, the darkest line represents optical probe B adjusted upwards by 3.0%, the light-colored line represents electrochemical probe B with an Agile filter (59s sampling rate and preset sample window), and the dashed lines represent the set point and upper / lower limits of the normal operating range).

[0125] Figure 58A and Figure 58B Examples of varying dextrose concentrations at different times are shown.

[0126] Figure 59 The pCO2 profile (y-axis) of culture media with different bubbling conditions (in mmHg) as of treatment time (days) according to an exemplary embodiment is shown. Medium pCO2 was selected as the midpoint control.

[0127] Figure 60 The observations are shown based on a model generated from a multivariate study of risk factors and reactions in mixed-mode chromatography (MMC) using Capto Adhere resin, according to an exemplary embodiment.

[0128] Figure 61The observations are shown from a model generated by a multivariate study of risk factors and responses of MMC using PPA HyperCel resin according to an exemplary embodiment. Detailed Implementation

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

[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 product's titer, batch size, yield, or quality. The applicant has discovered that, compared to serum-free media ("non-PS media") containing very little or no polyamines ("ornithine, putrescine, spermine, and spermidine"), the addition of polyamines ("PS media") to a medium ("PS media") improves viable cell density, cell doubling time, and dupilumab protein production in cell cultures.

[0131] Conventional methods for collecting and purifying protein drug products rely on a series of filtration and chromatographic steps that can be limited in their ability to handle increased titers and corresponding batch sizes, protein concentrations, and drug product viscosities while maintaining purity and quality standards using equipment of similar size. For example, when moving drug products with high protein concentrations, pumps previously used to move the drug products may become ineffective due to the increased viscosity levels. Techniques have been developed to reduce sample viscosity, such as adding arginine, a hydrophobic salt. However, adding arginine during collection and purification, particularly during concentration and percolation, can result in the loss of an unknown amount of arginine in subsequent processing steps, which must then be measured and compensated for for use in further downstream steps, and may increase variability in the final drug product. Therefore, there is a need for systems and methods for collecting high-titer antibody drug products while maintaining product consistency, high yield, and quality.

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

[0133] Dupilumab is a human monoclonal immunoglobulin G4 (IgG4) antibody, expressed and prepared from Chinese hamster ovary cells (CHO-K1) targeting 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 α (IL-4Rα) subunit shared by the IL-4 and IL-13 receptor complex. Dupilumab inhibits IL-4 signaling via type I receptors and IL-4 and IL-13 signaling via type II receptors. Blocking IL4Rα with dupilumab inhibits the inflammatory response induced by IL-4 and IL-13 cytokines, including the release of pro-inflammatory cytokines, chemokines, nitric oxide, and IgE. By blocking the specific 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 subsequently received marketing authorization in the European Union on September 28, 2017, and in Japan on January 19, 2018, followed by marketing authorizations in several 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 sinusitis with nasal polyps (CRSwNP), eosinophilic esophagitis (EoE), chronic spontaneous urticaria (CSU), and nodular prurigo (PN). Additionally, subcutaneous dupilumab is currently being developed for the treatment of patients with type 2 inflammatory diseases, including Alzheimer's disease (AD) in pediatric patients (6 months to <6 years old), asthma in pediatric patients (6 months to <6 years old), chronic obstructive pulmonary disease (COPD), allergic fungal sinusitis (AFRS), chronic sinusitis without nasal polyps (CRSsNP), allergies (e.g., grass allergy, peanut allergy, and dairy product allergy), bullous pemphigoid, hand and foot atopic dermatitis, cold urticaria, chronic inducible urticaria, ulcerative colitis, chronic pruritus of unknown cause, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, bronchiectasis, alopecia areata, and allergic rhinitis.

[0136] To meet the high demand for dupilumab, a new manufacturing process has been developed, as described further below. For example, the new process allows for the production and purification of dupilumab formulation active pharmaceutical ingredients (FDS) at scales of 10,000L and 25,000L, respectively.

[0137] Description of terms

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

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

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

[0141] This disclosure provides a serum-free culture medium suitable for culturing cells and producing biopharmaceutical raw materials. "Serium-free" refers to a cell culture medium that does not contain animal serum (such as fetal bovine serum). The serum-free culture medium may contain ≤7.5 g / L of hydrolysate, such as soybean hydrolysate. This disclosure also provides a chemically defined culture medium (CDM) that is not only serum-free but also free of hydrolysate.

[0142] "No hydrolysate" applies to cell culture media that do not contain exogenous protein hydrolysates, such as animal or plant protein hydrolysates, including, for example, peptone, tryptone, etc.

[0143] “Cell culture” or “culture” refers to the growth and reproduction of cells outside a multicellular organism or tissue. The culture conditions suitable for mammalian cells are known in the field. See, for example, *Animal cell culture: A Practical Approach*, ed., D. Rickwood, Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or simultaneously attached to a solid matrix. Fluidized bed bioreactors, hollow fiber bioreactors, roller flasks, flasks, or stirred tank bioreactors, with or without microcarriers, and operating in batch, batch-feed, 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 intervals during culture. For example, the culture can be fed once daily, every other day, every three days, or when the concentration of a particular culture medium component falls outside the desired range.

[0144] As used herein, the term "chemically defined medium" or "chemically defined media" (both abbreviated as "CDM") refers to a synthetic growth medium in which the identity and concentration of all components are known. Chemically defined media do not contain bacteria, yeast, animal or plant extracts, animal serum, or plasma, but may contain components derived from individual plants or animals (e.g., proteins, peptides, etc.). Chemically defined media may contain inorganic salts required to support growth, such as phosphates and sulfates. The carbon source is known and is typically a sugar, such as glucose, lactose, galactose, etc., or other compounds, such as glycerol, lactate, acetate, etc. While some chemically defined media also use phosphates as buffers, other buffers may be used, such as sodium bicarbonate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), citrate, triethanolamine, etc. Examples of commercially available culture media with defined chemical compositions include (but are not limited to) various Dulbecco's Modified Eagle's (DME) media (Sigma-Aldrich Co.; SAFC Biosciences, Inc.), Ham nutrient mixtures (Sigma-Aldrich Co.; SAFC Biosciences, Inc.), various EX-CELL media (Sigma-Aldrich Co.; SAFC Biosciences, Inc.), various IS CHO-CD media (FUJIFILM Irvine Scientific), and combinations thereof. Methods for preparing culture media with defined chemical compositions are known in the art, for example, U.S. Patent Nos. 6,171,825 and 6,936,441, WO2007 / 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 a cell into which a recombinant expression vector encoding a protein of interest has been introduced. It should be understood that such terms are intended not only to refer to a particular individual cell but also to the offspring of such cells. Such offspring may actually differ from the parent cell due to mutations or environmental influences, but are still included within the scope of the term "host cell" as used herein. In one embodiment, the host cell includes prokaryotic and eukaryotic cells selected from any living organism. In one aspect, eukaryotic cells include protists, fungi, plant, and animal cells. In another aspect, the host cell includes eukaryotic cells, such as plant and / or animal cells. Cells may be mammalian cells, fish cells, insect cells, amphibian cells, or avian cells. In a particular aspect, the host cell is a mammalian cell. A wide variety of mammalian cell lines suitable for growth in cultures are available from the American Type Culture Collection (Manassas, Virginia) and other repositories and commercial suppliers. Cells that can be used in the methods of this invention include (but are not limited to) MK2.7 cells, PER-C6 cells, Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCCCCL-61) (Chasin et al., 1986, Somatic Cell and Molecular Genetics, 12:555-556; Kolkekar et al., 1997, Biochemistry, 36:10901-10909; and WO 01 / 92337A2), dihydrofolate reductase-negative CHO cells (CHO / -DHFR, Urlaub and Chasin, 1980, Proceedings of the National Academy of Sciences of the United States of America, 77:4216), and dp12.CHO cells (US Patent No. 5,721,121); monkey kidney cells (CV1, ATCCCL-61). CCL-70); monkey kidney CV1 cells (COS cells, COS-7, ATCC CRL-1651) transformed with SV40;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 their strains (e.g., human embryonic kidney cells (e.g., HEK293 cells, or HEK293 cells subcloned for growth in suspension culture, Graham et al., 1977, Journal of General Virology, 36:59); juvenile hamster kidney cells (BHK, ATCC CCL-10); mouse setelli cells). Cells (TM4, Mather, 1980, Reproductive Biology, 23:243-251); Human cervical cancer cells (HeLa (HELA), ATCC CCL-2); Canine kidney cells (MDCK, ATCC CCL-34); Human lung cells (W138, ATCCCCCL-75); Human hepatocellular carcinoma cells (HEP-G2, HB 8065); Mouse mammary tumor cells (MMT 060562, ATCCCCCL-51); Buffalo rat hepatocytes (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals of the New York Academy of Sciences, 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, progeny 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 Fibroblasts from any tissue or organ (including but not limited to the heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphoid tissue (lymph nodes, adenoids, tonsils, bone marrow, and blood), and spleen), as well as fibroblasts and fibroblast-like cell lines (e.g., TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinated 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 cells, etc.), and fibroblasts and fibroblast-like cell lines (e.g., TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinated 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 cells, etc.). 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, and 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, strain 2071 (mouse L) cells, LM strain (mouse L) cells, L-MTK (mouse L) cells, NCTC strains 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntac cells, SIRC cells, C; IIThe cells used in this invention are CHO cells, HEK293 cells, BHK cells, or derivatives thereof, or any other cell type known to those skilled in the art. In some aspects, the cells used in this 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 be unrelated to the desired protein of interest. Host cell proteins can be process-related impurities, which may originate from the manufacturing process and can include three main categories: those derived from the cell matrix, those derived from cell cultures, and those derived 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 cultures include (but are not limited to) inducers, antibiotics, serum, and other culture medium components. Impurities derived downstream include (but are not limited to) enzymes, chemical and biochemical treatment agents (e.g., cyanogen bromide, guanidine, oxidants, and reductants), inorganic salts (e.g., heavy metals, arsenic, non-metallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leaching materials.

[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 flows (or enters) a stationary liquid or solid phase due to the differential distribution of its components. Non-limiting examples of liquid chromatography include reversed-phase liquid chromatography, ion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode chromatography. In some aspects, a sample containing at least one digest of a protein or peptide of interest may undergo any of the aforementioned chromatographic methods or a combination thereof. Analytes separated by chromatography will be characterized by a unique residence time, reflecting the rate at which the analyte moves through the chromatographic column. Chromatograms can be used to compare analytes, plotting residence time on one axis and the measured signal on another axis, where the measured signal may be generated by, for example, UV detection or fluorescence detection.

[0148] As used herein, the term "mass spectrometer" includes devices capable of identifying specific molecular species and measuring their precise mass. The term is intended to include any molecular detector by which polypeptides or peptides can be characterized. A mass spectrometer may comprise three main components: an ion source, a mass analyzer, and a detector. The ion source functions to generate gaseous ions. Analyte atoms, molecules, or clusters can be transferred to the gas phase and ionized simultaneously (e.g., 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 analysis" includes techniques in which structural information about sample molecules is obtained through the use of multi-stage 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 a first mass selection step. MS / MS or MS 2 This can be achieved by first selecting and separating precursor ions (MS). 1 This process involves fragmenting the ions to extract meaningful information. Tandem MS has been successfully implemented using a wide variety of analyzer combinations. The choice of which analyzers to combine for a particular 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 temporal tandem, but hybrids also exist where temporal tandem analyzers are located in space or coupled to spatial tandem analyzers. A spatial tandem mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-captured mass analyzers. Specific m / z separation functions can be designed to select ions in one part of the instrument, dissociate them in an intermediate region, and then transfer the product ions to another analyzer for m / z separation and data acquisition. In temporal tandem, mass spectrometer ions generated in the ion source can be trapped, separated, fragmented, and m / z separated within the same physical device.

[0150] Peptides identified by mass spectrometry can be used as representatives of intact proteins and their post-translational modifications or other modifications. They can be used for protein characterization by correlating experimental and theoretical MS / MS data, the latter generated from potential 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, locating post-translational modifications, identifying post-translational modifications, or performing comparability analysis, or a combination thereof.

[0151] In some exemplary aspects, mass spectrometry can be used for nanoelectrospray or nanospray. As used herein, the term "nanoelectrospray" or "nanospray" refers to electrospray ionization at extremely low solvent flow rates, typically hundreds of nanoliters per minute or less of sample solution without the use of external solvent delivery. Electrospray delivery devices that form nanosprays can use static or dynamic nanoelectrospray emitters. Static nanoelectrospray emitters perform continuous analysis of small sample (analyte) solution volumes over an extended period. Dynamic nanoelectrospray emitters use capillary columns and solvent delivery systems to chromatographically separate mixtures prior to analysis by mass spectrometry.

[0152] In some exemplary embodiments, mass spectrometry analysis may be performed under natural conditions. As used herein, the term “natural conditions” may include mass spectrometry analysis performed under conditions that preserve non-covalent interactions in the analyte. For a detailed review of natural 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, such as in a FASTA format file. Relevant protein sequences may be derived from the cDNA sequences of the species under study. Public databases available for searching relevant protein sequences include those 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 for 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 include 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 the production, processing, and / or purification of proteins. For example, downstream process technologies include the use of, for instance, affinity chromatography, ion exchange chromatography (such as anion or cation exchange chromatography), hydrophobic interaction chromatography, or displacement chromatography to separate protein products.

[0155] As used herein, the term "ultrafiltration" or "UF" can refer to a membrane filtration process similar to reverse osmosis, using hydrostatic pressure to force water through a semi-permeable membrane. Ultrafiltration is described in detail in Leos J. Zeman and Andrew L. Zydney, Microfiltration and Ultrafiltration: Principles and Applications (1996), the entire teachings of which are incorporated herein by reference. Filters with pore sizes less than 0.1 μm can be used for ultrafiltration. By employing 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 downstream of the filter.

[0156] As used herein, “diafiltration” or “DF” can include the use of an ultrafiltration apparatus 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 ionic and / or pH environments. Trace solutes can be removed most effectively by adding the solvent to the solution being ultrafiltered at a rate approximately equal to the ultrafiltration rate. This washes away trace species from the solution at a constant volume. In some exemplary embodiments of this disclosure, the diafiltration step can be used, for example, to exchange various buffer solutions used in conjunction with this disclosure prior to chromatography or other manufacturing steps, and to remove impurities from protein formulations.

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

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

[0159] The term "treat" or "treatment" refers to a therapeutic measure that reverses, stabilizes, or eliminates an unwanted disease or condition (such as atopic dermatitis (eczema), eosinophilic or oral steroid-dependent asthma, chronic sinusitis with nasal polyps (CRSwNP), eosinophilic esophagitis (EoE), inflammation), for example by reducing, stabilizing, or eliminating one or more symptoms or signs of such disease or condition to any clinically measurable degree. For example, in the case of asthma, it helps prevent severe asthma attacks (exacerbations), improves breathing, and helps reduce the amount of oral corticosteroids required.

[0160] As used herein, the term “critical quality attribute” (CQA) is used to describe the physical, chemical, biological, or microbiological characteristics or features that should be within appropriate limits, ranges, or distributions to ensure the quality of the desired product. CQA may include, for example, post-translational modifications.

[0161] As used herein, “virus filtering” 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 The filter, from Sartorius. CPV or HF, or Ultipor DV20 or DV50 from Pall Corporation. TM Or Pegasus TM Prime filters. It will be obvious to any technician in the field that selecting the right filter will achieve the desired filtration performance.

[0162] For biopharmaceuticals, the implementation of stable and flexible upstream processes is desirable. Effective upstream processes enable the desired production and scale-up of the protein of interest.

[0163] As used herein, a “sample” may be obtained from any step of a biological process, such as cell culture medium (CCF), collected cell culture medium (HCCF), any step of a downstream processing, active pharmaceutical ingredient (DS), or pharmaceutical product (DP) containing the final formulation. In some specific exemplary embodiments, the sample may be selected from any step of a downstream process, such as clarification, chromatographic production, or filtration.

[0164] As used herein, the terms "protein alkylating agent" or "alkylation agent" refer to a reagent used to alkylate certain free amino acid residues in a protein. Non-limiting examples of protein alkylating agents include iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylcis-butene diimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine, or combinations thereof.

[0165] As used herein, “protein denaturing” or “denaturation” can refer to the process by which the three-dimensional shape of a molecule is altered from its native state. Protein denaturation can be performed using a protein denaturing agent. Non-limiting examples of protein denaturing agents include heat, high or low pH, reducing agents (such as DTT, SDS), or exposure to a dissociating agent. Several dissociating agents can be used as protein denaturing agents. Dissociating a solute increases the entropy of a system by interfering with intramolecular interactions mediated by non-covalent forces (such as hydrogen bonds, van der Waals forces, and hydrophobic interactions). Non-limiting examples of dissociating agents include butanol, ethanol, carboxylic acid chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate (SDS), thiourea, N-lauroyl sarcosine, urea, and their salts. In some exemplary embodiments, the denaturing agent can be used in the mobile phase of a chromatographic analysis. In some exemplary embodiments, the denaturing agent 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 in a protein. Several methods exist for digesting proteins in a sample using appropriate hydrolytic agents, such as enzymatic or non-enzymatic digestion. The digestion of proteins into constituent peptides produces "peptide digests," which can be further analyzed using peptide localization analysis.

[0167] As used herein, the term "hydrolyzing agent" refers to any one or a combination of many different reagents capable of protein digestion. Non-limiting examples of hydrolyzing agents capable of enzymatic digestion include proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, Aspergillus pepsin I, LysN protease (Lys-N), LysC endopeptidase (Lys-C), endopeptidase Asp-N (Asp-N), endopeptidase Arg-C (Arg-C), endopeptidase Glu-C (Glu-C), or outer membrane protein T (OmpT) from Streptococcus pyogenes, immunoglobulin degrading enzyme (IdeS), thermophilic proteases, papain, streptomycin, V8 protease, or biologically active fragments or homologs thereof, or combinations thereof. Non-limiting examples of hydrolysants capable of non-enzymatic digestion include the use of high temperature, microwave, ultrasound, high pressure, infrared radiation, solvents (non-limiting examples being 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 teachings of which are incorporated herein by reference). One or a combination of hydrolysants can cleave peptide bonds in proteins or polypeptides in a sequence-specific manner, resulting in a predictable set of shorter peptides. The ratio of hydrolysant to protein and the time required for digestion can be appropriately selected to achieve optimal protein digestion. When the enzyme:substrate ratio is inappropriately high, the corresponding high digestion rate will result in insufficient time for mass spectrometry analysis of peptides, 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 a reagent used to reduce disulfide bonds in proteins. Non-limiting examples of protein reducing agents used to reduce proteins include dithiothreitol (DTT), β-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl), or combinations thereof.

[0169] As used herein, the term "protein" or "protein of interest" may include any amino acid polymer having covalently linked amide bonds. A protein comprises one or more amino acid polymer chains, which are generally referred to in the art as "peptides." A "peptide" is a polymer consisting of amino acid residues linked via peptide bonds, naturally occurring related structural variants thereof, and non-naturally occurring synthetic analogs. A "synthetic peptide or polypeptide" refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides may be synthesized, for example, using an automated peptide synthesizer. Various solid-phase peptide synthesis methods are known to those skilled in the art. A protein may contain one or more polypeptides to form a single-function biomolecule. In another exemplary aspect, a protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, etc. The protein of interest may include any of the following: biotherapeutic proteins, recombinant proteins for research or therapy, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, and human antibodies. 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 of 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 includes modifications, adducts, and other covalently linked portions. These modifications, adducts, and portions include, for example, avidin, streptavidin, biotin, polysaccharides (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAG tags, maltose-binding protein (MBP), chitosan-binding protein (CBP), glutathione S-transferase (GST), myc-epitopes, fluorescent labels, and other dyes.Proteins can be classified based on their composition and solubility and can therefore include simple proteins, such as globular proteins and fibrous proteins; conjugated proteins, such as nucleoproteins, glycoproteins, mucins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary and secondary derived proteins.

[0170] As used herein, the term "recombinant protein" refers to a protein resulting from the transcription and translation of a gene carried on a recombinant expression vector adapted to a host cell. In some exemplary embodiments, the recombinant protein may be an antibody, such as a chimeric antibody, a humanized antibody, or a fully human antibody. In some exemplary embodiments, the recombinant protein may be an isotype antibody selected from the group consisting of IgG, IgM, IgA1, IgA2, IgD, and IgE. In some 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 an immunoglobulin molecule comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) linked by disulfide bonds, as well as its multimers (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 contains 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 contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In various embodiments of this disclosure, the FR of the anti-large-ET-1 antibody (or its antigen-binding portion) may be identical to a human germline sequence or may be naturally or artificially modified. The common amino acid sequence may be defined based on a side-by-side analysis of two or more CDRs. As used herein, the term "antibody" also includes the antigen-binding fragment of a complete antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen-binding fragment of an antibody may be derived from, for example, a complete antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and optionally constant domains of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or may be synthetic. DNA can be sequenced and manipulated chemically or through molecular biology techniques, such as arranging one or more variable and / or constant domains into a suitable configuration, or introducing codons, generating cysteine ​​residues, modifying, adding or deleting amino acids, etc.

[0172] As used herein, an "antibody fragment" includes a portion of a complete antibody, such as the antigen-binding region or variable region of an antibody. Examples of antibody fragments include (but are not limited to) Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv bifunctional antibodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining regions (CDRs), as well as trifunctional antibodies, tetrafunctional antibodies, linear antibodies, and single-chain antibody molecules. An Fv fragment is a combination of variable regions of the immunoglobulin heavy and light chains, and a scFv protein is a recombinant single-chain polypeptide molecule in which the variable regions of the immunoglobulin light and heavy chains are linked by peptide linkers. In some exemplary embodiments, the antibody fragment contains a sufficient amino acid sequence of the parent antibody such that it is 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 a complete antibody and / or recombinantly by a gene encoding a partial antibody sequence. Alternatively, antibody fragments can be produced wholly or partially synthetically. Antibody fragments may optionally comprise single-chain antibody fragments. Alternatively, antibody fragments may comprise multiple chains linked together, for example, by disulfide bonds. Antibody fragments may optionally comprise multi-molecular complexes. Functional antibody fragments typically contain at least about 50 amino acids and more typically at least about 200 amino acids.

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

[0174] As used herein, “protein pharmaceutical product,” “biologic product,” or “biologic therapeutic agent” includes an active ingredient that may be wholly or partially biological in nature. In one aspect, a protein pharmaceutical product may comprise peptides, proteins, antibodies, antigens, peptide-drug conjugates, antibody-drug conjugates, protein-drug conjugates, cells, tissues, or combinations thereof. In another aspect, a protein pharmaceutical product may comprise recombinant, engineered, modified, mutated, or truncated forms of peptides, proteins, antibodies, antigens, vaccines, peptide-drug conjugates, antibody-drug conjugates, protein-drug conjugates, cells, tissues, or combinations thereof.

[0175] Exemplary anti-IL-4Rα antibody

[0176] As used in this paper, dupilumab (IgG4 isotype) is a covalent heterotetramer composed of two human heavy chains linked by disulfide bonds, each heavy chain covalently linked to a human κ 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 a hapten in solution. Each heavy chain contains a single N-linked glycosylation site (Asn). 302 The antibody, based on the primary sequence (without N-linked glycosylation), has a molecular weight of 146,897.0 Da, considering the formation of 16 disulfide bonds and the removal of Lys from the C-terminus of each heavy chain. 452 The variable domains of the heavy and light chains combine to form the complementarity-determining region (CDR) for binding dupilumab to its target interleukin-4 receptor α (IL-4Rα). Figure 1 The diagram shows a schematic representation of dupilumab, including the location of the N-linked glycosylation site and the disulfide bond structure.

[0177] The manufacturing process described herein can be used to produce human antibodies or antigen-binding fragments thereof that specifically bind to IL-4R (such as IL-4Rα) and contain three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO:1. The antibody or antigen-binding fragment may contain three light chain CDRs (LCVR1, LCVR2, and LCVR3) within a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:2. Methods and techniques for identifying CDRs within the 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 the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, 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 methods. See, for example, 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., Proceedings of the National Academy of Sciences 86:9268-9272 (1989). Public databases can also be used to identify CDR sequences within antibodies.

[0178] In some embodiments, the antibody or its antigen-binding fragment comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of the heavy and light chain variable region amino acid sequence pairs (HCVR / LCVR) from SEQ ID NO:1 and 2.

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

[0180] In some embodiments, the antibody or its antigen-binding fragment comprises the HCVR / LCVR amino acid sequence pairs of SEQ ID NO:1 and 2.

[0181] In some embodiments, the antibody is dupilumab, which contains the HCVR / LCVR amino acid sequence pairs of SEQ ID NO:1 and 2.

[0182] In some embodiments, the antibody sequence is dupilumab, which contains the heavy chain / light chain amino acid sequence pairs of SEQ ID NO: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 - 至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 some embodiments, the manufacturing method can be used to produce an antibody or an 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-68; SCB-VL-44 / SCB-VH-69 / SCB-VL-49 / SCB-VH-92; SCB-VL-44 / SCB-VH-69 / SCB-VL-49 / SCB-VH-92; SCB-VL-44 / SCB-VH-69 / SCB-VL-49 / SCB-VL-49 / SCB-VL-92; SCB-VL-4 ...49 / SCB-VL-49 / SCB-VL-49 / SCB-VL-49 / SCB-V 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 some embodiments, the antibody or its antigen-binding fragment comprises an LCVR / HCVR sequence pair of SCB-VL-44 / SCB-VH-92.

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

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

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

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

[0209] In some embodiments, the antibody or its antigen-binding fragment comprises: an HCVR comprising the HCDR1 sequence of SCB-92-HCDR1, the HCDR2 sequence of SCB-92-HCDR2, and the HCDR3 sequence of SCB-92-HCDR3; and an LCVR comprising the LCDR1 sequence of SCB-55-LCDR1, the LCDR2 sequence of SCB-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 some embodiments, the manufacturing method can be used to produce an antibody or an antigen-binding fragment thereof comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR) sequence pair selected from the group consisting of MEDI-1-VL / MEDI-1-VH to MEDI-42-VL / MEDI-42-VH (LCVR / HCVR).

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

[0219] In some embodiments, the antibody or its antigen-binding fragment comprises: an 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 an 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 some embodiments, the antibody or its antigen-binding fragment comprises an LCVR / HCVR sequence pair of AJOU-90-VL / AJOU-83-VH.

[0231] In some embodiments, the antibody or its antigen-binding fragment comprises: an 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 an 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 some embodiments, the antibody or antigen-binding fragment thereof disclosed herein comprises a light chain variable region (LCVR) and heavy chain variable region (HCVR) sequence pair (LCVR / HCVR) selected from the group consisting of sequences listed in Table 4, consisting of 11 / 3, 27 / 19, 43 / 35, 59 / 51, 75 / 67, 91 / 83, 107 / 99, 123 / 115, 155 / 147 and 171 / 163.

[0239] The antibodies listed in Table 4 below are described in more detail in U.S. Patent Nos. 7,605,237 and 7,608,693, which are incorporated herein by reference in their entirety for all purposes.

[0240] Table 4

[0241]

[0242]

[0243]

[0244] It should be understood that this disclosure is not limited to any of the aforementioned proteins, proteins of interest, antibodies, cell culture media, host cell proteins, protein alkylating agents, protein denaturing agents, protein reducing agents, digestive enzymes, treatments, processes, samples, surfactants, cleaning agents, chromatographic methods, filtration methods, mass spectrometers, databases, bioinformatics tools, pH, temperature, or concentration, and any protein, protein of interest, antibody, cell culture media, host cell protein, protein alkylating agent, protein denaturing agent, protein reducing agent, digestive enzyme, treatment, process, sample, surfactant, cleaning agent, chromatographic methods, filtration methods, mass spectrometers, databases, bioinformatics tools, pH, temperature, or concentration can be selected by any suitable means.

[0245] This disclosure will be more fully understood by referring to the following embodiments. However, these embodiments should not be construed as limiting the scope of the invention.

[0246] Cell culture medium and additives

[0247] Unfortunately, removing serum and reducing or eliminating hydrolysates from cell culture media while minimizing batch-to-batch variability and facilitating downstream processing steps weakens cell growth, reduces survival rates, and decreases protein expression. Therefore, serum-free and low- or even hydrolysate-free culture media with defined chemical compositions require additional components to improve cell growth and protein production. The cell culture media of this 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, etc., according to 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 combinations thereof (“PS medium”) to improve cell growth, cell viability, and recombinant protein production.

[0249] In one aspect, the PS medium contains concentrations (expressed in micromoles per 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, and 587. Ornithine at concentrations of 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 625, 630, 635, 640, 645, 650, 700, 750, 800, 850, or 900 ± 14 μM. In one aspect, PS medium contains ornithine at concentrations between approximately 0.09 and 0.9 mM.

[0250] In one embodiment, in addition to including ornithine or putrescine, the culture medium also contains 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 contains about 174 μM ± 26 μM of nucleoside. In one embodiment, the culture medium contains purine derivatives at cumulative concentrations 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. In one embodiment, the culture medium contains about 106 μM ± 5 μM of purine derivatives. Purine derivatives include hypoxanthine, as well as nucleosides adenosine and guanosine. In one embodiment, the culture medium contains pyrimidine derivatives at cumulative concentrations of at least 30 μM, at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, or at least 65 μM. In one embodiment, the culture medium contains about 68 μM ± 5 μM of pyrimidine derivatives. Pyrimidine derivatives include the nucleosides thymidine, uridine, and cytidine. In one particular embodiment, the culture medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

[0252] In one embodiment, in addition to ornithine or putrescine, the culture medium contains amino acids 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 additive" during cell culture, such as during protein production. Therefore, in some embodiments, such as in methods of culturing cells or producing the protein of interest, the culture medium may be supplemented with glutamine as a point-of-use additive. In one such embodiment, glutamine is added in amounts less than about 40 mM, less than about 35 mM, less than about 30 mM, less than about 25 mM, less than about 20 mM, less than about 15 mM, less than about 10 mM, less than about 8 mM, less than about 7 mM, less than about 6 mM, less than about 5 mM, less than about 4 mM, less than about 3 mM, or less than about 2.5 mM. In one embodiment, the amount of glutamine in the culture medium supplemented with glutamine is about 2 mM ± 0.5 mM.

[0253] In one embodiment, in addition to ornithine or a combination of ornithine and putrescine, the culture medium also contains amino acids with nonpolar side groups at concentrations 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 contains about 30 mM of amino acids with nonpolar side groups. In one embodiment, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, or at least 41% of the total amount of amino acids contained in the culture medium on a molar basis are amino acids with nonpolar side groups. In one embodiment, about 42 mol% ± 1 mol% of the amino acids in the culture medium are amino acids with nonpolar side groups. Amino acids with nonpolar 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 of amino acids listed 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 glutamic acid 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 ornithine or a combination of ornithine and putrescine, the culture medium also contains amino acids with uncharged polar side groups at concentrations 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 contains about 22 mM of amino acids with uncharged polar side groups. In another embodiment, the culture medium contains about 12 mM of amino acids with uncharged polar side groups. In one embodiment, of the total amount of amino acids contained in the culture medium, approximately 14% to 46%, approximately 15% to 45%, approximately 16% to 44%, approximately 17% to 43%, approximately 18% to 42%, approximately 19% to 41%, approximately 20% to 40%, approximately 21% to 39%, approximately 22% to 38%, approximately 23% to 37%, approximately 24% to 36%, approximately 25% to 35%, approximately 26% to 34%, approximately 27% to 33%, approximately 28% to 32%, approximately 29% to 31%, or approximately 30% are amino acids with uncharged polar side groups. In one embodiment, approximately 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 ornithine or a combination of ornithine and putrescine, the culture medium also contains, at concentrations 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 of an amino acid (e.g., an acidic amino acid) with a negative charge at pH 6. In one embodiment, the culture medium contains about 9 mM of acidic amino acids. In another embodiment, the culture medium contains 9 mM ± 1 mM of acidic amino acids. In one embodiment, about 8% to 18%, about 9% to 17%, about 10% to 16%, about 11% to 15%, about 12% to 14%, or about 13% of the total amino acids contained in the culture medium on a molar basis are acidic amino acids. In one embodiment, about 12.6 mol% ± 1 mol% of the amino acids in the culture medium are acidic amino acids. Acidic amino acids include aspartic acid and glutamic acid.

[0258] In one embodiment, in addition to ornithine or a combination of ornithine and putrescine, the culture medium also contains at least 3.5 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, or at least 11 mM of amino acids (e.g., basic amino acids) with a positive charge at pH 6. In one embodiment, the culture medium contains about 11 mM of basic amino acids. In one embodiment, the culture medium contains about 11.42 mM ± 1 mM of basic amino acids. In one embodiment, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15% of the total amount of amino acids contained in the culture medium (on a molar basis) 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, approximately 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 ornithine or a combination of ornithine and putrescine, the culture medium also contains about 30 mM of nonpolar amino acids, about 22 mM of uncharged polar amino acids, about 9 mM of acidic amino acids, and about 11 mM of 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 ornithine or a combination of ornithine and putrescine, the culture medium also contains micromolar amounts of fatty acids (or fatty acid derivatives) and tocopherol. In one embodiment, the fatty acids include one or more of the following: linoleic acid, linolenic acid, lipoic acid, oleic acid, palmitic acid, stearic acid, arachidonic acid, arachidonic acid, lauric acid, behenic acid, capric acid, dodecanoic acid, hexanoic acid, teicosanoic acid, myristic acid, and caprylic acid. In one embodiment, the culture medium contains tocopherol, linoleic acid, and lipoic acid.

[0261] In one embodiment, the culture medium further comprises a mixture of vitamins at 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 vitamin mixture comprises one or more of the following: D-biotin, choline chloride, folic acid, inositol, nicotinamide, pyridoxine HCl, D-pantothenic acid (hemicalcium), riboflavin, thiamine HCl, vitamin B12, etc. In one embodiment, the vitamin mixture comprises all of D-biotin, choline chloride, folic acid, inositol, nicotinamide, pyridoxine HCl, D-pantothenic acid (hemicalcium), riboflavin, thiamine HCl, and vitamin B12.

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

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

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

[0265] In one embodiment, the culture step lasts for about 10 to 18 days. In another embodiment, the culture 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 contains recombinant growth factors, osmotic weight molar concentration adjusters, pH buffers, glutamine, and cell protectants.

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

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

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

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

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

[0272] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium contains recombinant growth factors, osmotic weight molar concentration adjusters, pH buffers, glutamine, methotrexate, and cell protectants.

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

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

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

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

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

[0278] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium contains recombinant growth factors, osmotic weight molar concentration adjusters, pH buffers, glutamine, methotrexate, and cell protectants.

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

[0280] In one aspect, a batch-feed production method for dupilumab includes culturing Chinese hamster ovary (CHO) cells containing nucleic acids encoding said dupilumab in a cell culture production medium at scale, wherein sodium phosphate in said medium is supplemented on days 2 and 4 or on days 0, 2, 4, 6 and / or 8 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, such that the titer of said dupilumab in said cell culture production medium is about 5 g / L, about 6 g / L, about 7 g / L or about 8 g / L on day 10. In another 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 contains recombinant growth factors, osmotic weight molar concentration adjusters, pH buffers, glutamine, methotrexate, and cell protectants.

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

[0283] In one embodiment, the sodium phosphate in the culture medium is supplemented on days 2 and 4, or on days 0, 2, 4, 6, and / or 8, 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.

[0284] In one aspect, a batch-feed production method for dupilumab includes culturing Chinese hamster ovary (CHO) cells containing nucleic acids encoding said dupilumab in a cell culture production medium at scale, wherein sodium phosphate in said medium is supplemented on days 2 and 4 or on days 0, 2, 4, 6 and 8 to concentrations 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, respectively, wherein tyrosine in said medium is supplemented on day 3 at a concentration of about 2 g / L, and wherein insulin in said medium is supplemented on days 2 and 4 at a concentration of about 7.5 mg / L, such that the titer of said dupilumab in said cell culture production medium is at least 5 g / L on day 10.

[0285] In one aspect, a batch-feed production method for dupilumab includes culturing Chinese hamster ovary (CHO) cells containing nucleic acids encoding said dupilumab in a cell culture production medium at scale, wherein sodium phosphate in said medium is supplemented on days 2 and 4 or on days 0, 2, 4, 6 and 8 to concentrations 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, respectively, wherein tyrosine in said medium is supplemented on days 3 and 7 at a concentration of about 1 g / L, and wherein insulin in said medium is supplemented on days 2 and 4 at a concentration of about 7.5 mg / L, such that the titer of said dupilumab in said cell culture production medium is at least 5 g / L on day 10.

[0286] In one embodiment, the cell culture production medium is a serum-free medium. In some embodiments, the serum-free medium contains recombinant growth factors, osmotic weight molar concentration adjusters, pH buffers, glutamine, methotrexate, and cell protectants.

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

[0288] In one embodiment, the sodium phosphate in the culture medium is supplemented on days 2 and 4, or on days 0, 2, 4, 6, and 8, 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.

[0289] In one implementation, the culture lasts for approximately 10 days.

[0290] Various embodiments of the culture medium of the present invention include any combination of the embodiments described above, comprising a chemically defined, hydrolysate-free, serum-free culture medium containing a specified amount of ornithine or putrescine, and particularly containing: (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 may be added to the PS culture medium.

[0291] The applicant envisions that in the practice of this invention, any one or more or combinations of a variety of basal culture media may be used, to which ornithine or a combination of ornithine and putrescine may be added. Basal culture media are generally known in the art, and particularly include Eagle MEME (minimum essential medium) (Eagle, Science, 1955, 112(3168):501-504), Ham F12 (Ham, Proceedings of the National Academy of Sciences of the United States of America, 1965, 53:288-293), F-12K medium, Dalberg medium, Dalberg modified Eagle medium (Proceedings of the National Academy of Sciences of the United States of America, August 1952; 38(8):747-752), 1:1 DMEM / Ham F12, Trowell medium. T8, A2 medium (Holmes and Wolf, Biophysical and Biochemical Cell Biology, 1961, 10: 389-401), Waymouth medium (Davidson and Waymouth, Journal of Biochemistry, 1945, 39(2): 188-199), Williams E medium (Williams et al., Experimental Cell Research, 1971, 69: 105 and ff), RPMI 1640 (Moore et al., Journal of the American Medical Association, 1967, 199: 519-524), MCDB 104 / 110 medium (Bettger et al., Proceedings of the National Academy of Sciences, 1981, 78(9): 5588-5592), Ventrex HL-1 medium, albumin-globulin medium (Orr et al., Applied Microbiology (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 media, such as EX-CELL™ 300 series (JRH Biosciences, Lenexa, Kansas), protamine-zinc-insulin medium (Weiss et al., 1974, US 4,072,565), biotin-folic acid medium (Cartaya, 1978, US Re30,985), transferrin-fatty acid medium (Baker, 1982, US 4,560,655), transferrin-EGF medium (Hasegawa, 1982, US 4,615,977; Chesbeuf, 1984, US 4,786,599) and other culture medium alternatives (see Inlow, US 6,048,728; Drapeau, US 7,294,484; Mather, US 5,122,469; Furukawa, US 5,976,833; Chen, US 6,180,401; Chen, US 5,856,179; Etcheverry, US 5,705,364; Etcheverry, US 7,666,416; Ryll, US 6,528,286; Singh, US 6,924,124; Luan, US 7,429,491, etc.).

[0292] In a particular embodiment, the culture medium is chemically defined and, in addition to ornithine or a combination of ornithine and putrescine, contains: CaCl₂ 2H₂O; HEPES buffer, KCl; MgSO₄; NaCl; Na₂HPO₄ or other phosphates; pyruvate; L-alanine; L-arginine HCl; L-asparagine H₂O; L-aspartic acid; L-cysteine ​​HCl H₂O; 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; Nicotinamide; Pyridoxine HCl; D-Pantothenic Acid; Riboflavin; Thiamine HCl; Vitamin B12; Para-Aminobenzoic Acid; Ethanolamine HCl; Poloxamer 188; DL-α-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 osmotic weight mol concentration of the culture medium is 200 to 500, 250 to 400, 275 to 350, or about 300 mOsm. During cell growth in the culture medium, and especially after any feed according to a batch feeding protocol, the osmotic weight mol concentration of the culture can be increased to about 350, 400, 450, or up to 500 mOsm.

[0294] In some embodiments where the osmotic weight molality of the culture medium with defined composition is less than about 300, the osmotic weight molality is brought to about 300 by adding one or more salts in excess of a specified amount. In one embodiment, the osmotic weight molality is increased to the desired level by adding one or more osmotic agents selected from: 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 at a concentration greater than or equal to its concentration in the component already present in the culture medium with defined composition (e.g., sugars are added at a concentration greater than or equal to a concentration specified for the sugar component).

[0295] This disclosure provides a cell culture comprising a cell line expressing a protein of interest in an OS medium as described above. In one embodiment, the cell culture contains insulin, which may be added to the medium as a point-of-use component or may be included in the medium formulation. In one embodiment, the cell line comprises cells capable of producing a biotherapeutic protein. Examples of cell lines conventionally used for producing protein biotherapeutic agents include, in particular, 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 implementation, the cell line is a CHO cell line, or one or more of several specific CHO cell variants optimized for large-scale protein production, such as CHO-K1.

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

[0297] In some implementations, the cell culture medium is supplemented with "point-of-use additives," also known as additives, point-of-use components, or point-of-use chemicals, during cell growth or protein production. Point-of-use additives include 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, and testosterone. In some cases, growth factors may be non-natural chemicals that promote cell proliferation or protein production, such as tetrahydrofolate (THF) and methotrexate. Non-limiting examples of protein and peptide growth factors include angiopoietin, 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), hepatocellular carcinoma-derived growth factor (HDGF), insulin, and insulin-like growth factor (IGGF). F), migration-stimulating factors, myosin (GDF-8), nerve growth factor (NGF) and other neurotrophic factors, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor α (TGF-α), transforming growth factor β (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 added 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 added additives may also be included in the culture medium formulation of some embodiments.

[0298] The buffer is generally known in the art. This invention is not limited to any particular buffer, and anyone skilled in the art can choose an appropriate buffer or buffer system for use with a specific cell line producing a specific protein. In one embodiment, the addendum buffer used is NaHCO3. In another embodiment, the addendum buffer used contains NaHCO3. In yet another embodiment, the buffer is HEPES.

[0299] Energy sources suitable for use point additives in cell cultures are also well known in the art. Without limitation, in one embodiment, the energy source for the use point additive is glucose. Given the specific and particular requirements of a particular cell line and the 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 cell culture and protein production techniques. Tetrasodium EDTA dihydrate and citrate are two commonly used chelating agents in the field, but other chelating agents may be used in the practice of this invention. In one embodiment, tetrasodium EDTA dihydrate is used as the additive chelating agent. In another embodiment, citrate, such as Na3C6H5O7, is used as the additive chelating agent.

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

[0302] Other additives used 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 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 additives: 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 implementation, the culture medium is replenished at intervals during cell culture according to a batch feeding process. Batch feeding culture is generally known in the art and is used to optimize protein production (see YM Huang et al., Biotechnol Prog., Sep-October 2010; 26(5):1400-10).

[0305] Cell viability, viable cell density, and cell multiplication were improved compared to cells grown in media without ornithine or putrescine. Regarding cell viability, cells grown in PS medium exhibited a viability at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or at least 3 times higher than that of similar or identical cells grown in non-PS medium.

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

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

[0308] Protein production

[0309] In addition to the chemically defined PS medium and the method of culturing cells in the PS medium, this disclosure also provides a method for producing proteins, such as therapeutically effective antibodies or other biopharmaceutical raw materials, in cells cultured in the PS medium.

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

[0311] In some embodiments, the protein production yield or titer (which can be expressed in grams of protein product per liter of 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 (the 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 or tetrafunctional antibody, a Fab fragment or an 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 moiety and another domain. In some embodiments, the Fc moiety comprises a hinge region, followed by the CH2 and CH3 domains of IgG.

[0314] This invention is not limited to any specific type of cell for protein production. Examples of cell types suitable for protein production include mammalian cells, primate cells, insect cells, avian cells, bacterial cells, and yeast cells. Cells may be stem cells or recombinant cells transformed with a vector for recombinant gene expression, or cells transfected with a virus for producing viral products. Cells may contain a recombinant heteropolynucleotide construct encoding the protein of interest. The construct may be a free organism or may be an element physically integrated into the cell's genome. Cells may also produce the protein of interest without encoding it on a heteropolynucleotide construct. In other words, the cell may naturally encode the protein of interest, such as antibody-producing B cells. Cells may also be primary cells, such as chicken embryonic cells, or primary cell lines. Examples of suitable cell types 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 a CHO lec mutant.

[0315] In one embodiment, the cell is a CHO cell that ectopically expresses a protein. In one embodiment, the protein comprises immunoglobulin heavy chain regions, such as CH1, CH2, or CH3 regions. In one embodiment, the protein comprises human or rodent immunoglobulin CH2 and CH3 regions. In one embodiment, the protein comprises human or rodent immunoglobulin CH1, CH2, and CH3 regions. In one embodiment, the protein comprises a hinge region and CH1, CH2, and CH3 regions. In some embodiments, the protein comprises immunoglobulin heavy chain variable domains. In some embodiments, the protein comprises immunoglobulin light chain variable domains. In some embodiments, the protein comprises both immunoglobulin heavy chain variable domains and immunoglobulin light chain variable domains. 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 approximately 1,000 liters to 25,000 liters or greater. Protein production can be controlled using one or more of several methods, such as temperature variation or chemical induction. The growth phase can occur at higher temperatures than the production phase. For example, the growth phase can occur at a first temperature of approximately 35°C to 38°C, and the production phase can occur at a second temperature of approximately 29°C to 37°C, optionally at approximately 30°C to 36°C or approximately 30°C to 34°C. Additionally, chemical inducers for protein production, such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylene diacetamide (HMBA), can be added simultaneously with, before, or after temperature variation. If the inducer is added after temperature variation, it can be added from one hour to five days after the temperature variation, or one to two days after the temperature variation. Cell cultures can be produced as a continuous feed culture system, such as in a chemostat (see C. Altamirano et al., Progress in Biotechnology, Nov-December 2001; 17(6):1032-41), or according to a batch feed process (Huang, 2010).

[0317] This invention relates to improving protein production via cell culture processes. Cell lines used in this invention can be genetically engineered to express peptides of commercial or scientific interest. Genetically engineered cell lines involve transfecting, transforming, or transducing cells with recombinant polynucleotide molecules, or otherwise altering them (e.g., through homologous recombination and gene activation, or fusion of recombinant cells with non-recombinant cells) to enable host cells to express the desired recombinant peptide. Methods and vectors for genetically engineering cells or cell lines to express the peptide of interest are well known to those skilled in the art; for example, various techniques are described in the following literature: Current Protocols in Molecular Biology, eds. Ausubel et al. (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 cultures are available from the U.S. Collection of Type Cultures (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), Journal of Biochemistry 263:6352-6362; McKinnon et al. (1991), Journal of Molecular Endocrinology 6:231-239; Wood et al. (1990), Journal of Immunology 145:3011-3016). The dihydrofolate reductase (DHFR) deficient mutant cell line (Urlaub et al. (1980), Proceedings of the National Academy of Sciences 77:4216-4220) DXBI 1 is the desired CHO host cell line because the highly efficient DHFR selectable and amplifiable gene expression system allows for high levels of recombinant protein expression in these cells (Kaufman RJ. (1990), Enzymatic Methods (MethEnzymol) 185:537-566).Furthermore, these cells are easily manipulated as attached or suspended cultures and exhibit relatively good genetic stability. CHO cells and their recombinantly expressed proteins have been extensively characterized and approved for commercial manufacturing. In some embodiments, the CHO cell lines are those described in U.S. Patent Application Publications Nos. 2010 / 0304436A1, 2009 / 0162901A1, and 2009 / 0137416A1, and U.S. Patents Nos. 7,455,988B2, 7,435,553B2, and 7,105,348B2.

[0318] This disclosure is not limited to the specific embodiments described herein, which are intended as illustrative examples of individual aspects or implementations of the invention. Functionally equivalent methods and components are within the scope of this invention. Various modifications to the invention, other than those described herein, will be readily apparent to those skilled in the art based on the foregoing description and drawings. Such modifications fall within the scope of this invention.

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

[0320] Seed expansion culture optimization and measurement

[0321] As described herein, an improved method has been developed to optimize the large-scale production of proteins in cell cultures by implementing a seed expansion culture process that forces and biases cells to increase the live cell density (VCD) in the large-scale production vessel. In U.S. Application No. 16 / 984,581 (incorporated by reference), entitled Metabolically Optimized Cell Culture, it has previously been observed that peak lactate consumption in seed expansion cultures can be used to improve the large-scale production of proteins. As described herein, a novel seed expansion culture process has been developed that further enhances cell metabolism and productivity by modulating the initial VCD and without affecting the observed lower peak lactate consumption, demonstrating a novel and distinct mechanism for enhancing protein production compared to the mechanism described in U.S. Application No. 16 / 984,581.

[0322] As is well known from the background art, protein production via cell cultures is typically carried out using a batch or batch feeding process. The early stages of inoculum growth after thawing vials involve culturing cells in seed-scaled cultures. Culture vessels include (but are not limited to) well plates, corrugated bags, T-flasks, shake flasks, stirred containers, rotary flasks, hollow fiber flasks, airlift bioreactors, etc. Suitable cell culture vessels are bioreactors. A bioreactor is any culture vessel manufactured or engineered to manipulate or control environmental conditions. Such culture vessels are well known in the field. Typically, cells are grown at an exponential growth rate, such as in a seed-scaled culture bioreactor, to gradually increase the size and / or volume of the cell population. After the cell mass has been scaled up proportionally through several bioreactor stages, the cells are transferred to a production bioreactor while the cells are still in the exponential growth (logarithmic phase) (Gambhir, A. et al., 2003, J Bioscience Bioeng 95(4):317-327). In batch cultures (e.g., seed cultures), cells entering the stationary phase beyond the logarithmic phase are generally considered undesirable. It has been recommended that cultures be passaged while they are in the logarithmic phase, before cells (e.g., attached cells) reach confluence, because contact inhibition or waste accumulation can inhibit cell growth, and for other reasons (Cell Culture Basics, Gibco / Invitrogen Online Handbook, www.invitrogen.com; Animal Cell Culture Guide, www.atcc.org).

[0323] After being transferred to fed-batch cultures, cells are cultured for a period of time while the composition of the medium is monitored and controlled to allow for the production of the protein or peptide of interest. Once a specific yield is reached, or cell viability, waste accumulation, or nutrient consumption determines that culture should be terminated, the produced protein or peptide is isolated. Efforts, particularly regarding reducing the output of metabolic waste (such as lactate accumulation) in cell cultures, have improved overall final protein droplet measurements. These efforts focus on controlling glucose or nutrient-limited batch feeding processes (see, for example, WO2004104186; U.S. Patent No. 8,192,951B2), improving cell culture conditions (e.g., U.S. Patent No. 7,390,660; Zagari et al., 2013, New Biotechnol., 30(2):238-45), or cell engineering, including targeting enzymes in the glycolysis 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, Journal of Biotechnology 131, 168-176).

[0324] Controlling cell feeding is used to pursue 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 nutrient deprivation and the fact that rapid changes in ammonia concentration, such as those seen in high-density batch feeding 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 suitable high density in batch feed and then intentionally induce an extended production steady-state period through, for example, temperature or pH changes (Quek et al., 2010, Metabolic Engineering (Metab Eng) 12(2):161-71. Digital Object Identifier: 10.1016 / j.ymben.2009.09.002. Electronic version 13 October 2009).

[0325] In some embodiments, the initial VCD in the seed expansion culture process is adjusted to enhance protein production during batch or staggered feeding processes. In some aspects, the range is between 1.7 × 10⁻⁶ at each step (from N-5 to N-1). 5 Cells / mL to 2.3 × 10⁻⁶ 5 Compared to the acceptable standard initial VCD of cells / mL, the initial VCD increased to approximately 3.5 × 10⁻⁶ cells / mL. 5 Cells / mL up to 5.43 × 10⁻⁶ 5 Cells / mL. In some respects, the initial VCD was approximately 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 times higher than that of surrogate initial VCD in acceptable standard seed expansion cultures.

[0326] In some embodiments, increasing the initial VCD in containers N-5 to N-1 does not change the final VCD in N-1 compared to standard seed expansion culture. In one aspect, optimized seed expansion culture causes an increase in the final VCD in containers N-5 to N-2 compared to standard seed expansion culture. In one aspect, the peak lactate observed in the final production container is not substantially different from that of standard seed expansion culture. In one aspect, optimized seed expansion culture results in a sustained increase in biomass. In one aspect, optimized seed expansion culture causes an increase in final titer (g / L) of 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 skilled in the art how to use optimized seeds to expand culture, and is not intended to limit the scope of the invention as regarded by the inventors.

[0328] Given the importance of VCD in seed culture expansion 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 cultures is based on offline analysis (Ritacco, Biotechnology Progress, 34(6):1407-1426, 2018). For example, the trypan blue rejection test (TBET) can visually distinguish between live and non-live cells using a manual hemocytometer or an automated bioanalyzer; however, offline determination does not comply with the regulatory framework of the U.S. Food and Drug Administration (FDA), which implements mechanisms for designing, analyzing, and controlling pharmaceutical manufacturing processes (e.g., process analysis techniques / (PAT)) by measuring critical process parameters (CPP) that affect critical quality attributes (CQA) online or in-line (Metze, Bioprocess and Biosystems Engineering, 43(2):193-205, 2020). Offline measurements also introduce a delay between sampling and the response to the process, and require more labor and resources.

[0329] It should be understood that online and / or inline methods and systems for measuring VCD are required to optimize the production of therapeutic proteins from cell cultures and to adhere to the regulatory framework for the implementation of methodological analytical techniques outlined by the FDA.

[0330] Dielectric spectroscopy is a promising technique for real-time analysis of cell culture and biomass properties. It is a non-destructive, non-invasive continuous process monitoring tool that can generate continuous process data for both suspensions and attached cell cultures. However, the implementation of biomass in-line monitoring techniques remains challenging due to the complexity of associated calibration processes, integration difficulties, and insufficient model accuracy and transferability (Carvell, Cell Technology, 50:35-48, 2006; Kell et al., Journal of Bioelectricity, 4(2):317-348, 1990). Therefore, this application provides the benefits of a dielectric spectroscopy system and method that can accurately determine in-line live cell density predictions during cell culture.

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

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

[0333] In one aspect, a wired sensor is used to measure the primary characteristics of cell cultures.

[0334] In one aspect, correlation equations are used to predict a second characteristic of a cell culture based on a first characteristic measured in the cell culture.

[0335] In one aspect, cell culture characteristics may include capacitance, viable cell density, capacitance, and / or conductivity.

[0336] In one embodiment, this disclosure provides a method for measuring the VCD of cells cultured in a bioreactor by applying an electric field to the cells cultured in the bioreactor, measuring the capacitance, and establishing a correlation between the measured capacitance and the live cell density.

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

[0338] In one aspect, offline assays are used to measure a second characteristic of a first cell culture.

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

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

[0341] In one aspect, correlation equations are derived using more than one cell line.

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

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

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

[0345] In one aspect, multivariate data analysis is used to generate correlation equations.

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

[0347] In one aspect, cell culture nutrients may include, for example, glucose, glutamine, glutamic acid, sodium, potassium, calcium, O2, or combinations thereof.

[0348] In one aspect, cell culture metabolites may include, for example, acetic acid, acetate, lactic acid, lactate, ammonia, ethanol, CO2, or combinations 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 repulsion tests), or combinations thereof. Cell density refers to the number of cells in a given volume of culture medium.

[0350] Example 23 is provided to describe to those skilled in the art how to use an improved capacitance probe to measure a VCD, and is not intended to limit the scope of the invention as regarded by the inventors.

[0351] Optimization of stirring, bubbling, and measurement in containers and bioreactors

[0352] Optimizing the initial volume, air bubbling, and agitation setpoints in containers and bioreactors is crucial because during the growth and production phases, cell cultures are exposed to shear stress from the agitator impeller and bubbling gas, which is necessary to maintain adequate oxygen delivery and dissolved carbon dioxide stripping to sustain cell health. These processes have been found to be optimized by using stepped or varying agitation and air bubbling rates. For example, stepped or varying agitation and air bubbling rates have been found to provide significant improvements in VCD (Voice Controlled Calibration). Dissolved oxygen and carbon dioxide can be controlled at the setpoint via bubbling in a cascaded control loop.

[0353] It has also been found that stepwise or altered agitation and air bubbling rates can reduce heterogeneity by decreasing the amount of acidic and basic species in the recombinant proteins of interest. These variants can arise from deamidation, sialylation, glycosylation, and fragmentation, which can alter the stability, activity, and potency of proteins containing the Fc moiety (the portion derived from the crystallizable fragment region of the antibody). Sissolak et al., J. Indust. Microbiol. Biotech. 46:1167-78 (2019). Basic variants can increase antibody binding to the Fc receptor. Hintersteiner et al., MABS8:1458-60 (2016).

[0354] Fc glycans can play roles in immunogenicity, biological activity, pharmacodynamics, and pharmacokinetics. (Reusch and Tejada, Glycobiol. 25:1325-34 (2015)). A possible phenomenon is called unglycosylated heavy chain (NGHC). NGHC variation can alter effector functions, such as opsonization. Opsonization involves the Fc moiety in ADCC (antibody-dependent cytotoxicity), ADCP (antibody-dependent 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 moiety.

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

[0356] In the other case, the initial stirring rate was about 75 rpm, which was then increased to 150 rpm and then to 225 rpm. In one exemplary embodiment, the air bubbling 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 days 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11. In one aspect, the initial bubbling rate is between 50 slpm and 75 slpm, and increases to approximately 225 slpm to 275 slpm on the second day. In another aspect, the bubbler has between 146 and 292 holes with a size between 0.5 mm and 2 mm, and may include, for example, bubblers having holes of 146 × 0.5 mm, 146 × 1.0 mm, 146 × 1.5 mm, 146 × 2.0 mm, 292 × 0.5 mm, 292 × 1.0 mm, 292 × 1.5 mm, and 292 × 2.0 mm.

[0357] In one aspect, the stirring rate and the air bubbling rate are increased on the same day. In the other aspect, the stirring rate and the air bubbling rate are increased on different days, wherein the stirring rate can be as follows: Figure 45 The results show an increase in daily feed rates.

[0358] In one exemplary embodiment, the initial capacity of the vessel or bioreactor can be optimized to reduce agitation-related surface effects. The initial operating capacity within the vessel or bioreactor is typically set at a level with sufficient clearance to allow for capacity adjustments, including subsequent feed. Unexpectedly, increasing the initial operating capacity has been found to increase both biomass and final titer. It has been found that increasing the operating capacity reduces agitation-related surface effects caused by the impeller in the bioreactor, including air cascade effects, and provides better control over dissolved oxygen concentrations.

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

[0360] Table 6

[0361] Unit operations Original volume (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 implementation, dissolved oxygen levels 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 a set time period. In one aspect, the bubbling rate can be configured to automatically adjust the bubbling rate based on the measured dissolved oxygen level to maintain a set level of dissolved oxygen, which can be constant, variable, or stepped as described above using cascaded control.

[0363] As described above, dissolved oxygen and carbon dioxide concentrations are critical process parameters that must be carefully controlled, necessitating optimized systems and methods for measuring dissolved gases. In one embodiment, an improved probe for measuring dissolved gases in large-scale containers and bioreactors used in the biopharmaceutical industry for culturing cells in the manufacture of therapeutic protein products is disclosed. An improved data processing method is derived by comparing data from two optical probes (referred to as optical probe A and optical probe B) with data from an electrochemical probe in a large-scale industrial bioreactor. As shown in Examples 19 and 21 below, a calibration method is generated to account for the observed offsets relative to the electrochemical probes between the two optical probes (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. Notably, the optical probes exhibit 100% offset elimination above the normal operating range observed on the electrochemical probes, and approximately twice the noise reduction (based on standard deviation comparison) compared to the electrochemical probes.

[0364] Additionally, as shown in Example 20, an improved signal processing method is generated, including filtering and smoothing data to power the chemical probe to reduce offset events identified as false alarms. In one aspect, signal processing is applied in real time.

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

[0366] In one embodiment, a container or bioreactor with reduced maintenance requirements is configured to measure dissolved oxygen using one or more optical probes. The bioreactor and container may also be configured to have one or more optical probes to measure other gases, such as carbon dioxide. In one aspect, data from the optical probes is processed based on a fixed offset. In one aspect, the offset is based on the correlation between data from the optical probes and the electrochemical probes. In another aspect, the offset is based on the correlation with the optical probes and known standards. In yet another aspect, data from the optical probes 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 container or bioreactor is configured to use one or more electrochemical probes, which have optimized data processing by filtering and smoothing the signals from the electrochemical probes. In one aspect, the sampling window for data is smoothed between a 10-minute window and a 33-minute window.

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

[0369] In one embodiment, cells are cultured under appropriate pCO2 conditions that control for antibody heterogeneity and for 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. Cells can be any suitable mammalian cell type, including CHO, BHK, HEK293, HeLa, human amniotic membrane, Per.C6, and Sp2 / 0 cells. While not bound by any theory, it is believed that increasing the CO2 content in the culture medium will cause an increase in intracellular CO2, which can induce charge and glycosylation variations. This effect is separate from any decrease in pH that may be due to the formation of carbonic acid.

[0370] Carbon dioxide concentration can be increased using CO2 bubbling or by reducing air bubbling. Lowering the pressure in a production bioreactor will reduce oxygen solubility; this, in turn, requires more bubbling to maintain the dissolved oxygen (DO) setpoint and an increased gas flow rate, which in turn lowers 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] Cells can be cultured for approximately 10 to 15 days. In one embodiment, cells can be cultured for approximately 14 days. During culture, pCO2 conditions can be between approximately 30 mmHg and approximately 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 measurable using a CO2 electrode.

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

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

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

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

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

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

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

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

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

[0381] Additional details are based on antibodies containing FcR proteins, such as those capable of binding to the IL-4 receptor, as further described in the following Example 18, 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 the acidic species and unglycosylated heavy chains of dupilumab.

[0382] In one exemplary embodiment, the glucose feeding strategy is also optimized to prevent high osmotic molality conditions. It has been found that glucose concentration affects osmotic conditions, which can affect cell culture performance. To improve cell culture conditions and performance, the dextran target is reduced to prevent high osmotic molality conditions. In one aspect, the dextran target is reduced by 10%, 20%, 30%, 40%, 50%, 60%, or 70%. In one aspect, the dextran target is gradually increased on day 2, day 3, day 4, day 5, or day 6. In one aspect, the dextran target is gradually increased and then decreased after day 0.5, day 1, day 1.5, day 2, day 2.5, or day 3. In one exemplary embodiment, the initial dextran target is between 5 g / L and 7 g / L, and the gradually increasing dextran target is between 7 g / L and 11 g / L. In one embodiment, the dextran target is gradually increased and gradually decreased once or more. Figure 58A and Figure 58B The example shows dextrose targets and their regulation over time.

[0383] In another embodiment, seed culture and production media and feed can be subjected to a 10-second High Temperature Short Time (HTST) treatment at 102°C. Alternatively, HTST treatments can be performed for 8, 9, 11, 12, 13, 14, or 15 seconds at temperatures of approximately 101°C, 102°C, 103°C, 104°C, 105°C, or approximately 106°C. HTST can serve as a rapid pasteurization of cell culture media and feed, and can be used in manufacturing as a viral barrier for upstream processing and to reduce the risk of exogenous factors, thereby producing equivalent process performance and product quality, including titers.

[0384] Centrifugation, depth and fine filtration

[0385] In some exemplary embodiments, a single recovery may include one or more centrifugation steps, including disc-stack centrifugation and deep, fine, and protective filtration, or direct deep and fine filtration without centrifugation, to separate the protein of interest from the host cell and accompanying cell debris. Figure 28 The diagram illustrates an exemplary overview of the use of depth filters, fine filters, and protective filters.

[0386] Samples can be centrifuged at, for example (but not limited to), rates from 7,000 × g to approximately 12,750 × g. It is recommended to fill the drum with 60% to 80% solids to calculate the discharge interval. The centrifuged liquid is then treated with in-line depth, fine, and protective filtration. Therefore, the filter flux varies with the centrifuge feed rate and the area of ​​the depth, fine, and protective filters. In some embodiments, a drum speed of 5550 rpm (7000 g) and a feed flow rate of approximately 1630 L / h can be used. Filter flux and discharge intervals 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 rotation speed can affect turbidity by altering the settling rate of particles of various sizes. In large-scale production scenarios, such centrifugation can be set to be performed linearly at a flow rate that achieves a turbidity level of, for example, 150 NTU in the resulting supernatant.

[0392] Cell culture media or centrifuged media may need to be adjusted to specific pH and conductivity in order to achieve the desired impurity removal and product recovery by a deep filtration step.

[0393] To reduce batch-to-batch variability, the turbidity of the centrifuged liquid should be measured at the midpoint between discharges (referred to as the drum midpoint (t = 1 / 2 discharge interval)) to determine whether adjustments to the drum speed and feed flow rate are necessary. 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 also been found that centrifugation performance cannot be predicted using linear extrapolation based solely on the δ concept because the amount of shear induced by centrifugation changes with drum speed and thus alters the particle size distribution.

[0394] To overcome these limitations, predictive profilers are generated to observe, for example... Figure 29 The factors and reactions reflected in it.

[0395] The obtained turbidity model was significant (p<0.0001), with R²adj of 0.95 and RMSE of 25.0 FNU. The primary and secondary effects of relative centrifugal force, the primary effect of flow rate, and the interaction between relative centrifugal force and flow rate were identified as significant factors. Flow rate had the greatest impact on the turbidity of the centrifuged liquid. Generally, when RCF is less than 8000 g, the turbidity of the centrifuged liquid varies only with the flow rate.

[0396] The centrifuged liquid can then be collected for further processing or filtered online via one or more depth filters for further clarification. The most commonly used depth filters for biological processing consist of diatomaceous earth, cellulose fibers, and a positively charged resin binder. Unlike membrane filters, depth filters retain particles throughout the porous filter media, allowing for the retention of particles larger and smaller than the pore size. Particle retention is believed to involve both size exclusion and adsorption via ionic, hydrophobic, and other mechanisms. Filter fouling mechanisms can include pore blockage, cake formation, and pore shrinkage. Non-limiting examples of depth filters that can be used in the context of this disclosure include Millistak+X0HC, F0HC, D0HC, C0HC, AlHC, B1HC depth filters (EMD Millistak), and 3M. TM Models 30 / 60ZA, 60 / 90ZA, VR05, VR07, and degreasing depth filters (3M Corp.). 0.2μm filters, such as Sartorius' 0.45 / 0.2μm Sartopore. TM Dual-layer or Millipore SHR or SHC filter cartridges are typically placed after the depth filter. The Emphaze AEX hybrid purifier multi-mechanism filter can also be used to filter material that has already undergone depth filtration. Other filters familiar to those skilled in the art may also be used.

[0397] Following deep filtration, fine filtration is used to ensure the removal of final solids before column chromatography. Fine filtration may include specially designed filters that enhance impurity absorption via ionic mechanisms and thus reduce the overall impurity content applied to the primary column chromatography step. Protective filtration following fine filtration is used solely for bioload control.

[0398] To accommodate high titers and increased protein production, the pore size of these fine and protective filters can range from 0.15 μm to 0.25 μm, and in some cases, can be 0.2 μm. For an average bioreactor culture of 10,000 L, the fine filter loading can be up to 255 L / m³. 2 Up to 270L / m 2 Within the range, and in one aspect, approximately 265 L / m 2 Up to 270L / m 2 (For example, 33.6m) 2 / 10,000L batch). Protective filter loading can be up to 700L / m³. 2 Up to 720L / m 2 Within the range.

[0399] Protein A chromatography

[0400] In some exemplary embodiments, it may be advantageous to perform affinity chromatography on a biological sample to produce the 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. Chromatographic materials also include those comprising, for example, a protein or a portion thereof capable of binding to or interacting with the protein of interest.

[0401] Affinity chromatography may involve placing a biological sample onto a column containing a suitable protein A resin. When used herein, the term "protein A" encompasses protein A recovered from its natural source, protein A produced synthetically (e.g., via peptide synthesis or recombinant technologies), and protein A with a retained binding affinity of C. H 2 / C H Variants of the ability of proteins in region 3. Protein A can also bind to human IgG molecules containing the IgG F(ab')2 fragment from the human VH3 gene family (Roben et al., Journal of Immunology 1995 154(12):6437-45). In some respects, protein A resins are suitable for the affinity-based generation and separation of multiple antibody isotypes by specifically interacting with the Fc portion of the molecule (if it has the region described).

[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, and rProtein A Sepharose from Cytiva; ProSep HC, ProSep Ultra, and ProSep Ultra Plus from EMD Millipore; MabCapture from Thermo Fisher Scientific; and Amsphere from JSR Life Sciences. TM A3.

[0403] Before loading the sample, the affinity column can be equilibrated with a suitable buffer. The pH for loading protein A 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 loading the sample, the column can be washed once or several times with a suitable wash buffer. The column can then be eluted with an appropriate elution buffer (e.g., glycine-HCl, acetic acid, or citric acid). Techniques familiar to those skilled in the art, such as a UV detector, can be used to monitor the eluent. The eluent fraction of interest can be collected and then prepared for further processing.

[0404] Protein A wash buffers can be selected based on their ability to disrupt protein-protein interactions (e.g., the interaction between the protein of interest and an impurity such as HCP) without disrupting the interaction between the protein of interest and the chromatographic material. Suitable wash buffers for HCP removal may contain, for example, salts (e.g., sodium salts such as sodium phosphate; potassium salts such as potassium sorbate; magnesium salts; salts containing hydrochloride such as guanidine hydrochloride, Tris-containing salts), surfactants (e.g., polysorbate 20, polysorbate 80), polar materials (e.g., isopropanol, ethanol), or amino acids (e.g., arginine). Suitable wash buffers for HCP removal may have a pH between about 5 and about 9, specifically about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9.

[0405] A suitable wash buffer can be selected based on the removal of a specific HCP, for example, by choosing the pH of the wash buffer corresponding to the pI of the relevant HCP. The wash buffer can be selected based on, for example, disrupting the electrostatic interaction between the relevant HCP and the protein of interest, the hydrophobic interaction between the relevant HCP and the protein of interest, or both. The specific properties of the relevant HCP can be used to inform the selection of buffer components as shown, for example, in Table 8. HCP profiling can be used to identify relevant HCPs, for example, by using mass spectrometry to identify and quantify HCPs associated with the protein of interest. A suitable wash buffer can be selected based on ensuring an acceptable yield of the protein of interest by minimizing disruption of the binding of the protein of interest to the chromatographic resin.

[0406] Table 8. Exemplary buffer components and pH values ​​that disrupt protein-protein interactions during affinity trapping.

[0407]

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

[0409] Using affinity wash buffers to reduce HCP content and / or HMW species can enable the achievement of corresponding drug substance quality with fewer downstream chromatographic steps, such as using protein A and AEX columns instead of CEX or HIC columns; or using a protein A column followed by a mixed-mode column and AEX column instead of CEX or HIC columns. If optimization via a single affinity capture wash buffer does not match the impurity content achievable with additional columns, an additional specific gravity can be obtained by using charged filter media during the filtration step. This is typically performed after the virus inactivation step, during which some pH adjustments have been made and filtration for specific conditions is easily incorporated into the downstream process. Filter media with multiple removal mechanisms are commercially available, including cationic, anionic, hydrophobic, and mixed-mode mechanisms. Because the charge density on the filter is typically lower 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 rates.

[0410] Low pH maintenance

[0411] In some exemplary embodiments, a single recovery process may also be a key step in reducing or inactivating viruses that may be present in biological samples. During the single recovery phase of production, any one or more of a variety of virus reduction / inactivation methods may be used, including heat inactivation (pasteurization), pH inactivation, buffer / cleaning agent treatment, UV and gamma irradiation, and the addition of certain chemical inactivating agents, such as β-propiolactone or, for example, copper phenotype as described in U.S. Patent No. 4,534,972, the entire teachings of which are incorporated herein by reference. In another aspect, virus-retaining filtration may also be used as a dedicated virus removal step and is discussed below.

[0412] In those exemplary embodiments employing virus reduction / inactivation, in one aspect, the biological sample can be adjusted as needed for use in other production steps. For example, after low-pH virus inactivation, the pH of the sample can be adjusted to approximately neutral pH, such as approximately 4.5 to approximately 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] Alternatively, the eluent can be inactivated by, for example, cleaning agents or low pH. A suitable cleaning agent concentration or pH (and time) can be selected to obtain the desired virus inactivation result.

[0414] In another aspect, after virus inactivation, the pH and / or conductivity of the eluent can be adjusted for use in subsequent production steps, including anion exchange chromatography, cation exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., protein A), and size exclusion chromatography.

[0415] Anion exchange chromatography

[0416] In some exemplary embodiments, the protein of interest is produced by performing at least one anion exchange (AEX) separation step on a biological sample. In one case, the anion exchange step may be performed after an affinity chromatography procedure (e.g., protein A affinity). In other cases, the anion exchange step may be performed before the affinity chromatography step. In some embodiments, AEX separation is the second operation of a four-unit chromatographic operation and is performed downstream of affinity capture and virus inactivation via a low pH maintenance operation. In other embodiments, the ion exchange step precedes AEX separation. Alternatively, another ion exchange procedure may follow AEX separation.

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

[0418] The use of anion exchange materials relative to cation exchange materials is partly based 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] During separation, the initial protein composition (biological sample) can be contacted with anion exchange material using any of a variety of techniques, such as batch production or chromatography.

[0420] In 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 is obtained. Biological samples can be contacted with the slurry to allow proteins to adsorb onto the anion exchange material. Solutions containing acidic substances that do not bind to the AEX material can be separated from the slurry by sedimentation and removal of the supernatant. One or more washing and / or elution steps can be performed on the slurry.

[0421] In chromatographic separation, a packed bed column is used to contain the chromatographic support material (resin or solid phase). A sample containing the protein of interest is loaded onto the specific column. The column can then be subjected to one or more washing steps using a suitable washing buffer. Components of the sample not yet adsorbed onto the resin may flow through the column. Components already adsorbed onto the resin can be eluted using a suitable elution buffer via differential elution.

[0422] In some embodiments, the amount of protein loaded on 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, or about 100 g / L. / L, approximately 105g / L, approximately 110g / L, approximately 115g / L, approximately 120g / L, approximately 125g / L, approximately 130g / L, approximately 135g / L, approximately 140g / L, approximately 145g / L, approximately 150g / L, approximately 155g / L, approximately 160g / L, approximately 165g / L, approximately 170g / L, approximately 175g / L, approximately 180g / L, approximately 185g / L, approximately 190g / L, approximately 195g / L, or approximately 200g / L.

[0423] In some embodiments, the sample may be neutralized before contact with the AEX material. The sample, for example, a virus-inactivated aggregate, may be neutralized to the following pH values: 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, and about 7.59. Approximately 7.60, approximately 7.61, approximately 7.62, approximately 7.63, approximately 7.64, approximately 7.65, approximately 7.66, approximately 7.67, approximately 7.68, approximately 7.69, approximately 7.70, approximately 7.75, approximately 7.80, approximately 7.85, approximately 7.90, approximately 7.95, approximately 8.00, approximately 8.05, approximately 8.10, approximately 8.15, approximately 8.20, approximately 8.25, or approximately 8.30.

[0424] In some embodiments, the sample may be further adjusted after neutralization and before contact with the AEX material. The sample, for example, a virus-inactivated aggregate, may be adjusted to a pH between approximately 3.00 mS / cm and approximately 6.00 mS / cm, between approximately 3.00 mS / cm and approximately 4.00 mS / cm, between approximately 3.40 mS / cm and approximately 3.60 mS / cm, approximately 3.00 mS / cm, approximately 3.10 mS / cm, approximately 3.20 mS / cm, approximately 3.30 mS / cm, approximately 3.40 mS / cm, approximately 3.50 mS / cm, approximately 3.60 mS / cm, approximately 3.70 mS / cm, approximately 3.80 mS / cm, approximately 3.90 mS / cm, approximately 4.00 mS / cm, and approximately 4. The following concentrations are possible: 10 mS / cm, approximately 4.20 mS / cm, approximately 4.30 mS / cm, approximately 4.40 mS / cm, approximately 4.50 mS / cm, approximately 4.60 mS / cm, approximately 4.70 mS / cm, approximately 4.80 mS / cm, approximately 4.90 mS / cm, approximately 5.00 mS / cm, approximately 5.10 mS / cm, approximately 5.20 mS / cm, approximately 5.30 mS / cm, approximately 5.40 mS / cm, approximately 5.50 mS / cm, approximately 5.60 mS / cm, approximately 5.70 mS / cm, approximately 5.80 mS / cm, approximately 5.90 mS / cm, or approximately 6.00 mS / cm. Samples can be adjusted using, for example, approximately 2 M sodium acetate, approximately 2 M Tris base, or combinations thereof.

[0425] In some embodiments, the AEX column undergoes a pre-equilibration step before contact with the sample. This pre-equilibration step replaces the strongly bound hydroxide ions on the AEX column and promotes faster equilibration. In some exemplary embodiments, the pre-equilibration buffer (or “mobile phase”) contains approximately 2M sodium chloride, WFI, or a combination thereof. In some exemplary embodiments, the linear velocity of the pre-equilibration buffer is between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, and is 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 equilibrated before contact with the sample and optionally after a pre-equilibration step. The equilibration step alters the pH and conductivity of the mobile phase to match the buffer excipients present in the loaded material (sample). In some embodiments, the equilibration buffer may contain about 50 mM Tris and about 60 mM acetate, with 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 equilibration 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, and is 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, and about... 7.60, approximately 7.61, approximately 7.62, approximately 7.63, approximately 7.64, approximately 7.65, approximately 7.66, approximately 7.67, approximately 7.68, approximately 7.69, approximately 7.70, approximately 7.75, approximately 7.80, approximately 7.85, approximately 7.90, approximately 7.95, approximately 8.00, approximately 8.05, approximately 8.10, approximately 8.15, approximately 8.20, approximately 8.25, or approximately 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, and is approximately 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, and about 4... 0.10 mS / cm, approximately 4.20 mS / cm, approximately 4.30 mS / cm, approximately 4.40 mS / cm, approximately 4.50 mS / cm, approximately 4.60 mS / cm, approximately 4.70 mS / cm, approximately 4.80 mS / cm, approximately 4.90 mS / cm, approximately 5.00 mS / cm, approximately 5.10 mS / cm, approximately 5.20 mS / cm, approximately 5.30 mS / cm, approximately 5.40 mS / cm, approximately 5.50 mS / cm, approximately 5.60 mS / cm, approximately 5.70 mS / cm, approximately 5.80 mS / cm, approximately 5.90 mS / cm, or approximately 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 on the AEX column (“AEX loading”, 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, and is 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, and about 15.0 g / L. Approximately 16.0 g / L, approximately 17.0 g / L, approximately 18.0 g / L, approximately 19.0 g / L, approximately 20.0 g / L, approximately 21.0 g / L, approximately 22.0 g / L, approximately 23.0 g / L, approximately 24.0 g / L, approximately 25.0 g / L, approximately 26.0 g / L, approximately 27.0 g / L, approximately 28.0 g / L, approximately 29.0 g / L, or approximately 30.0 g / L. In some exemplary embodiments, the linear speed of the AEX load 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 implementations, when the UV absorbance at 280 nm reaches 0.2 AU over a 5 mm flow path, approximately 1.2 column volumes of flow material are collected in the loading step. This is followed by a washing step to increase the yield.

[0429] In AEX chromatography, washing steps are typically performed using conditions similar to those used for loading, or alternatively by gradually or linearly decreasing the pH of the washing buffer and / or increasing its ionic strength / conductivity. In one aspect, the saline solution used for loading and washing buffers has a pH equal to or close to the isoelectric point (pI) of the protein of interest. Typically, the pH is about 0 to 2 units above or below the pI of the protein of interest, but it can be in the range of 0 to 0.5 units above or below. It can also be equal to the pI of the relevant protein.

[0430] In some embodiments, the washing 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 washing 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, and is approximately 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, and about... 7.60, approximately 7.61, approximately 7.62, approximately 7.63, approximately 7.64, approximately 7.65, approximately 7.66, approximately 7.67, approximately 7.68, approximately 7.69, approximately 7.70, approximately 7.75, approximately 7.80, approximately 7.85, approximately 7.90, approximately 7.95, approximately 8.00, approximately 8.05, approximately 8.10, approximately 8.15, approximately 8.20, approximately 8.25, or approximately 8.30. In some embodiments, the conductivity of the washing 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, and is approximately 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, and about 4... 0.10 mS / cm, approximately 4.20 mS / cm, approximately 4.30 mS / cm, approximately 4.40 mS / cm, approximately 4.50 mS / cm, approximately 4.60 mS / cm, approximately 4.70 mS / cm, approximately 4.80 mS / cm, approximately 4.90 mS / cm, approximately 5.00 mS / cm, approximately 5.10 mS / cm, approximately 5.20 mS / cm, approximately 5.30 mS / cm, approximately 5.40 mS / cm, approximately 5.50 mS / cm, approximately 5.60 mS / cm, approximately 5.70 mS / cm, approximately 5.80 mS / cm, approximately 5.90 mS / cm, or approximately 6.00 mS / cm.In some embodiments, the linear velocity of the washing 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 washing length is between about 3 column volumes (CV) and about 5 column volumes (CV), specifically 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 strip buffers. In some embodiments, the first strip buffer contains 2M sodium chloride (NaCl) and the second strip buffer contains 1N sodium hydroxide (NaOH). In some embodiments, the AEX column may be further immersed 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] Anionic reagents can be selected from the group consisting of acetate, chloride, formate, and combinations thereof. Cationic reagents can be selected from the group consisting of Tris, arginine, sodium, and combinations thereof. Buffer solutions can be selected from the following group: pyridine, piperazine, L-histidine, Bis-Tris, bisBis-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] Packed anion exchange columns, anion exchange membrane devices, anion exchange monomer devices, or depth filtration media can be operated in binding-elution, flow-through, or mixed modes, wherein proteins exhibit binding to the chromatographic material and can still be eluted from such material using the same or substantially similar buffer as the loading buffer.

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

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

[0437] In some implementations, the AEX step is performed in a negative mode (flow mode), in which negatively charged process-related impurities are adsorbed onto immobilized positively charged ligands, and the protein of interest flows through.

[0438] Non-limiting examples of anion exchange resins include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary ammonium (Q) groups. Other non-limiting examples include: Poros 50PI and Poros 50HQ, which are rigid polymeric beads having a backbone composed of cross-linked poly[styrene-divinylbenzene]; Poros 50XQ; Capto Q Impres and Capto DEAE, which are high-flow-rate 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 is a synthetic polymeric resin with tentacled ion exchangers; Sartobind PA nano, a salt-resistant chromatographic membrane with primary amine ligands; Sartobind Q nano, a strong anion exchange chromatographic membrane; CUNO BioCap, a ζ+ depth filtration medium composed of inorganic filter aids, refined cellulose, and ion exchange resins; XOHC, a depth filtration medium composed of inorganic filter aids, cellulose, and mixed cellulose esters; and Unosphere Q. In some embodiments, resins with relatively large pore sizes are selected to address the 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 batch is processed simultaneously or sequentially. In some embodiments, the batch may not be split after the AEX step.

[0440] Additives such as polyethylene glycol (PEG), detergents, amino acids, sugars, and liquid separators 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 the protein of interest. In some exemplary embodiments, the CEX step is performed in addition to the AEX step, either before or after the AEX step. In some embodiments, CEX is a third chromatographic unit operation in the purification process.

[0443] During cation exchange, a sample containing the protein of interest can be contacted with the cation exchange material using any of a variety of techniques (e.g., batch production or chromatography), as described above regarding AEX. Cation exchange packed bed chromatography is based on the ionic interaction between the bound entity (target protein or impurity) and functional groups immobilized on the chromatographic medium. Performance can vary with, for example, the mobile phase, elution conditions, functional groups, and resin backbone. In some embodiments, the specific objective 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 on 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, and is 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, the CEX column is pre-stripped before initiating a new separation to remove any bound impurities. In some embodiments, the pre-stripping buffer contains approximately 2M sodium chloride (NaCl). In some embodiments, approximately two column volumes of pre-stripping buffer are used. In some embodiments, the pre-stripping step is used only for the first cycle of the batch.

[0446] In some embodiments, the CEX column undergoes an equilibration step to alter the mobile phase pH and conductivity to facilitate adsorption. In some embodiments, the equilibration buffer contains approximately 40 mM sodium acetate, has a pH between approximately 5.90 and approximately 6.10, and a conductivity between approximately 2.00 mS / cm and approximately 4.00 mS / cm. In some embodiments, the equilibration buffer contains 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, and about 2. 20 mS / cm, approximately 2.30 mS / cm, approximately 2.40 mS / cm, approximately 2.50 mS / cm, approximately 2.60 mS / cm, approximately 2.70 mS / cm, approximately 2.80 mS / cm, approximately 2.90 mS / cm, approximately 3.00 mS / cm, approximately 3.10 mS / cm, approximately 3.20 mS / cm, approximately 3.30 mS / cm, approximately 3.40 mS / cm, approximately 3 0.50 mS / cm, approximately 3.60 mS / cm, approximately 3.70 mS / cm, approximately 3.80 mS / cm, approximately 3.90 mS / cm, approximately 4.00 mS / cm, approximately 4.10 mS / cm, approximately 4.20 mS / cm, approximately 4.30 mS / cm, approximately 4.40 mS / cm, approximately 4.50 mS / cm, approximately 4.60 mS / cm, approximately 4.70 mS / cm, approximately 4.80 mS / cm, approximately 4.90 mS / cm, approximately 5.00 mS / cm, approximately 5.10 mS / cm, approximately 5.20 mS / cm, approximately 5.30 mS / cm, approximately 5.40 mS / cm, approximately 5.50 mS / cm, approximately 5.60 mS / cm, approximately 5.70 mS / cm, approximately 5.80 mS / cm, approximately 5.90 mS / cm, or approximately 6.00 mS / cm. In some embodiments, approximately three column volumes of equilibration buffer are used.

[0447] The sample (CEX loading) can be conditioned before contact with CEX material. In some embodiments, the sample (e.g., AEX aggregate) is conditioned 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, about 2M acetic acid is used to conditioned the sample.

[0448] In some embodiments, the CEX column is subjected to a washing step to remove weakly bound impurities. In some embodiments, an equilibration buffer is also used as a washing buffer. In some embodiments, the washing buffer contains 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 washing buffer contains 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 washing 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, and about 2. 20 mS / cm, approximately 2.30 mS / cm, approximately 2.40 mS / cm, approximately 2.50 mS / cm, approximately 2.60 mS / cm, approximately 2.70 mS / cm, approximately 2.80 mS / cm, approximately 2.90 mS / cm, approximately 3.00 mS / cm, approximately 3.10 mS / cm, approximately 3.20 mS / cm, approximately 3.30 mS / cm, approximately 3.40 mS / cm, approximately 3 0.50 mS / cm, approximately 3.60 mS / cm, approximately 3.70 mS / cm, approximately 3.80 mS / cm, approximately 3.90 mS / cm, approximately 4.00 mS / cm, approximately 4.10 mS / cm, approximately 4.20 mS / cm, approximately 4.30 mS / cm, approximately 4.40 mS / cm, approximately 4.50 mS / cm, approximately 4.60 mS / cm, approximately 4.70 mS / cm, approximately 4.80 mS / cm, approximately 4.90 mS / cm, approximately 5.00 mS / cm, approximately 5.10 mS / cm, approximately 5.20 mS / cm, approximately 5.30 mS / cm, approximately 5.40 mS / cm, approximately 5.50 mS / cm, approximately 5.60 mS / cm, approximately 5.70 mS / cm, approximately 5.80 mS / cm, approximately 5.90 mS / cm, or approximately 6.00 mS / cm. In some embodiments, approximately two column volumes of wash buffer are used. In some embodiments, the wash buffer contains potassium sorbate.

[0449] Following the washing step, the proteins of interest can be eluted by an increase in conductivity. In some embodiments, CEX pooling begins with 0.2 AU using a 2 mm UV path and ends after approximately 5 column volumes (CV) of pooling. In some embodiments, the elution buffer contains approximately 20 mM Tris and approximately 120 mM sodium acetate, with a pH between approximately 5.90 and approximately 6.20, and a conductivity between approximately 9.00 mS / cm and approximately 11.00 mS / cm. In some embodiments, the elution buffer contains pH values ​​between approximately 5.70 and approximately 7.00, between approximately 5.90 and approximately 6.20, approximately 5.70, approximately 5.80, approximately 5.90, approximately 6.00, approximately 6.10, approximately 6.20, approximately 6.30, approximately 6.40, approximately 6.50, approximately 6.60, approximately 6.70, approximately 6.80, approximately 6.90, or approximately 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, and is about 7.00 mS / cm, about 7.10 mS / cm, about 7.20 mS / cm, and about 7.30 mS / cm. m, approximately 7.40 mS / cm, approximately 7.50 mS / cm, approximately 7.60 mS / cm, approximately 7.70 mS / cm, approximately 7.80 mS / cm, approximately 7.90 mS / cm, approximately 8.00 mS / cm, approximately 8.10 mS / cm, approximately 8.20 mS / cm, approximately 8.30 mS / cm, approximately 8.40 mS / cm, approximately 8.50 mS / cm, approximately 8.60 m S / cm, approximately 8.70 mS / cm, approximately 8.80 mS / cm, approximately 8.90 mS / cm, approximately 9.00 mS / cm, approximately 9.10 mS / cm, approximately 9.20 mS / cm, approximately 9.30 mS / cm, approximately 9.40 mS / cm, approximately 9.50 mS / cm, approximately 9.60 mS / cm, approximately 9.70 mS / cm, approximately 9.80 mS / cm, approximately 9. 90 mS / cm, approximately 10.00 mS / cm, approximately 10.10 mS / cm, approximately 10.20 mS / cm, approximately 10.30 mS / cm, approximately 10.40 mS / cm, approximately 10.50 mS / cm, approximately 10.60 mS / cm, approximately 10.70 mS / cm, approximately 10.80 mS / cm, approximately 10.90 mS / cm, or approximately 11.00 mS / cm. In some embodiments, approximately 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 further recycling or storage. In some embodiments, the first stripping buffer contains about 2M sodium chloride (NaCl) and the second stripping buffer contains about 1N sodium hydroxide (NaOH).

[0451] In some embodiments, each stage of separation, excluding elution, is performed at a linear velocity between approximately 100 and approximately 300 cm / hr, between approximately 150 and approximately 250 cm / hr, approximately 100 cm / hr, approximately 110 cm / hr, approximately 120 cm / hr, approximately 130 cm / hr, approximately 140 cm / hr, approximately 150 cm / hr, approximately 160 cm / hr, approximately 170 cm / hr, approximately 180 cm / hr, approximately 190 cm / hr, approximately 200 cm / hr, approximately 210 cm / hr, approximately 220 cm / hr, approximately 230 cm / hr, approximately 240 cm / hr, approximately 250 cm / hr, approximately 260 cm / hr, approximately 270 cm / hr, approximately 280 cm / hr, approximately 290 cm / hr, or approximately 300 cm / hr. In an exemplary embodiment, each stage of separation, excluding elution, is performed at a linear velocity of 200 cm / hr. In an exemplary embodiment, the elution step is performed at a linear speed 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] A saline solution can be used as both a loading and washing buffer for samples with a pH lower than the isoelectric point (pI) of the protein of interest. In one case, the pH is approximately 0 to 5 units lower than the protein's pI. In another case, it is in the range of 1 to 2 units lower than the protein's pI. In yet another case, it is in the range of 1 to 1.5 units lower than the protein's pI.

[0454] In some exemplary embodiments, the concentration of the anionic reagent 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 reagent 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 the CEX method include (but are not limited to) Tris formate, Tris acetate, ammonium sulfate, sodium chloride, and sodium sulfate.

[0455] In some exemplary embodiments, the conductivity and pH of the saline solution are adjusted by increasing or decreasing the concentration of the cation reagent. In one aspect, the concentration of the cation reagent 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 the cation reagent may be selected from the group consisting of sodium, Tris, triethylamine, ammonium, arginine, and combinations thereof.

[0456] Packed cation exchange columns or cation exchange membrane devices can operate in bind-elution, flow-through, or mixed modes, where products exhibit binding or interaction with chromatographic materials but can still be eluted from such materials using the same or substantially similar buffer as the loading buffer (details of these modes are outlined above). In some embodiments, the CEX step is performed in a positive (bind-elution) mode, where positively charged proteins of interest and impurities are adsorbed onto a stationary phase with a negative charge. The proteins of interest are then 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). Other cationic materials include (but are not limited to): Capto SPImpRes, high-flow-rate agarose beads; CaptoS ImpAct; F-grade CM Hyper D, coated ceramic beads permeated with functionalized hydrogel (250 to 400 ionic groups μeq / mL); Eshmuno S, a hydrophilic polyvinyl ether basic matrix with an ionic capacity of 50 to 100 μM / mL; Nuvia C Prime, a hydrophobic cation exchange medium composed of a macroporous, highly cross-linked hydrophilic polymer matrix with a capacity of 55 to 75 με / mL; Nuvia S, an UNOspher basic matrix with ionic groups of 90 to 150 με / mL; Poros HS, rigid polymer beads with a backbone composed of cross-linked poly(styrene-divinylbenzene); Poros XS, rigid polymer beads with a backbone composed of cross-linked poly(styrene-divinylbenzene); Toyo Pearl Giga Cap CM 650M, a polymeric basic bead with an ion capacity of 0.225 meq / mL; Toyo Pearl Giga Cap S650M, a polymeric basic bead; Toyo Pearl MX TRP, a polymeric basic bead; and EMD SE Hicap. It should be noted that CEX chromatography can be used with the MM resin 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 the cationic substrate.

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

[0459] Mixed mode chromatography

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

[0461] Capto Adhere is a column resin suitable for mixed-mode separation. Capto Adhere is a strong anion exchanger with multimodal functionality. Its basic matrix is ​​highly cross-linked agarose with a ligand (N-phenylmethyl-N-methylethanolamine), exhibiting different functionalities for interactions, such as ionic interactions, hydrogen bonding, and hydrophobic interactions. In some respects, resins for mixed-mode separation are selected from PPA-HyperCel and HEA-HyperCel. The basic matrix of PPA-HyperCel and HEA-HyperCel is highly porous cross-linked cellulose. Their ligands are amphetamine and hexylamine, respectively. Amphetamine and hexylamine provide different selectivity and hydrophobicity options for protein separation. Other mixed-mode chromatographic carriers include (but are not limited to) Capto MMC, MEP-HyperCel, MBI HyperCel, CMM HyperCel, Capto Adhere ImpRes, Capto Core 700, Nuvia C Prime, Toyo Pearl MX Trp 650M, and... HCX. In some respects, mixed-mode chromatographic resins contain ligands coupled to organic or inorganic supports, sometimes indicated directly or via spacers from a basic matrix. Supports can be in particulate form, such as substantially spherical particles, monomers, filters, membranes, surfaces, capillaries, etc. In some respects, supports are prepared from natural polymers, such as cross-linked carbohydrate materials, such as agarose, agar, cellulose, polydextrose, polyglucosamine, konjac, carrageenan, galvanic acid, alginate, etc. To obtain high adsorption capacity, supports can be porous, and ligands are subsequently 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: Acta Biochim Biophysica Sinica 79(2), 393-398 (1964), the entire contents of which are incorporated herein by reference). Alternatively, the support can be prepared from synthetic polymers, such as crosslinked synthetic polymers, for example styrene or styrene derivatives, divinylbenzene, acrylamide, acrylates, methacrylates, vinyl esters, vinylamides, etc. 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 teachings of which are incorporated herein by reference). Porous natural or synthetic polymer supports are also available from commercial sources, such as Cytiva, Uppsala, Sweden. MMC can be operated in either flow-through or binding-elution mode, depending on the resin used; for example, AEX / HIC blends are preferably operated in flow-through mode, while CEX / HIC blends are preferably operated in binding-elution mode.

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

[0463] The method disclosed herein can also be implemented in a continuous chromatographic mode. In this mode, at least two columns (referred to as a "first" column and a "second" column) are used. In some embodiments, this continuous chromatographic mode can be performed such that the eluted fractions and / or stripped fractions can subsequently or simultaneously be loaded onto the second column (with or without dilution).

[0464] In one embodiment, the medium for continuous mode can be one of various chromatographic resins, monomer media, membrane adsorbent media, or depth filtration media having side-connected hydrophobic and anion exchange functional groups.

[0465] In some exemplary embodiments, MMC is a second chromatographic separation in the process following affinity capture chromatography and optionally preceding the AEX step. In other embodiments, MMC is a third chromatographic separation in the process following affinity capture chromatography and the AEX step. In some embodiments, the method of this disclosure may include an MMC step instead of a CEX or HIC step. In some embodiments, the process for producing recombinant proteins (such as dupilumab) includes two, three, or four chromatographic modes, including affinity capture chromatography, MMC, and optionally 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 flow-through mode or in binding and elution mode. MMC can be operated based on a culture plate type (e.g., a 96-well plate type or a robotic column chromatography type).

[0466] In some embodiments, the MMC column undergoes an equilibration step to alter the mobile phase pH and conductivity to facilitate adsorption. In some embodiments, the equilibration buffer contains approximately 100 mM NaCl at a pH of approximately 5. In some embodiments, the equilibration buffer contains pH values ​​between approximately 4.50 and approximately 9.00, between approximately 4.50 and approximately 8.00, between approximately 4.50 and approximately 5.50, between approximately 5.00 and approximately 6.00, approximately 4.50, approximately 4.60, approximately 4.70, approximately 4.80, approximately 4.90, approximately 5.00, approximately 5.10, approximately 5.20, approximately 5.30, approximately 5.40, approximately 5.50, approximately 5.60, approximately 5.70, approximately 5.80, approximately 5.90, approximately 6.00, and approximately 6.10. pH values ​​of approximately 6.20, 6.30, 6.40, 6.50, 6.60, 6.70, 6.80, 6.90, 7.00, 7.10, 7.20, 7.30, 7.40, 7.50, 7.60, 7.70, 7.80, 7.90, 8.00, 8.10, 8.20, 8.30, 8.40, 8.50, 8.60, 8.70, 8.80, 8.90, or 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; and about 120 mM. The equilibration buffer contains approximately 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, or 500 mM NaCl. In some embodiments, the equilibration buffer contains between approximately 0 mM and approximately 100 mM NaCl. In some embodiments, the equilibration buffer contains citrate or arginine.

[0468] In some embodiments, the washing buffer may contain any of the pH, NaCl, concentration, or buffer salt of the aforementioned equilibration buffer.

[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 ... 0 g / L, about 105 g / L, about 110 g / L, about 115 g / L, about 120 g / L, about 125 g / L, about 130 g / L, about 135 g / L, about 140 g / L, about 145 g / L, about 150 g / L, about 155 g / L, about 160 g / L, about 165 g / L, about 170 g / L, about 175 g / L, about 180 g / L, about 185 g / L, about 190 g / L, about 195 g / L, or about 200 g / L. In some embodiments, the amount of protein loaded on the MMC resin can be between about 10 g / L and about 80 g / L, about 10 g / L, about 15 g / L, about 20 g / L, about 25 g / L, about 30 g / L, about 35 g / L, about 40 g / L, about 45 g / L, about 50 g / L, about 55 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, or about 80 g / L.

[0470] In some embodiments, the MMC operates in a binding and elution mode, and further includes the use of an elution buffer. In some embodiments, the elution buffer contains concentrations 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, and about 6.10. pH values ​​of approximately 6.20, 6.30, 6.40, 6.50, 6.60, 6.70, 6.80, 6.90, 7.00, 7.10, 7.20, 7.30, 7.40, 7.50, 7.60, 7.70, 7.80, 7.90, 8.00, 8.10, 8.20, 8.30, 8.40, 8.50, 8.60, 8.70, 8.80, 8.90, or 9.00.

[0471] In some embodiments, the elution buffer comprises approximately 0 mM to approximately 500 mM, approximately 100 mM to approximately 250 mM, approximately 100 mM to approximately 150 mM, approximately 0 mM, approximately 5 mM, approximately 10 mM, approximately 15 mM, approximately 20 mM, approximately 25 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM, approximately 55 mM, approximately 60 mM, approximately 65 mM, approximately 70 mM, approximately 75 mM, approximately 80 mM, approximately 85 mM, approximately 90 mM, and approximately 95 mM. NaCl at concentrations of approximately 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, or 500 mM. In some embodiments, the elution buffer contains citrate or arginine.

[0472] Hydrophobic interaction chromatography

[0473] The methods disclosed herein may include at least one hydrophobic interaction chromatography (HIC) step on a biological sample containing the protein of interest. During separation, the biological sample is contacted with HIC material, for example using batch production 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, HIC separation is the fourth and final chromatographic separation in the process and is performed downstream of the CEX step.

[0474] In some implementations, a specific objective of the HIC step may be, for example, to reduce the content of impurities associated with the HCP or HMW products, including PLBD2. The PLBD2 content in the HIC loading may be present in the range of 100-300 ppm and may 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 removal of PLBD2 using HIC is described in more detail in U.S. Patent No. 10,774,141, which is incorporated herein by reference in its entirety for all purposes.

[0475] In some implementations, a batch of samples can be split into two sub-batches, which are processed individually via a HIC step and subsequent steps, such as virus interception filtration and UF / DF, before recombination, for example, before third-stage concentration.

[0476] While ion-exchange chromatography relies on the local charge of the protein of interest for selective separation, hydrophobic interaction chromatography (HIC) utilizes the hydrophobic properties of proteins to achieve selective separation. HIC resins are typically functionalized with aromatic or aliphatic hydrocarbon ligands. Hydrophobic groups on or within the protein interact with hydrophobic groups on the chromatographic resin or membrane. Generally, under suitable conditions, the stronger the hydrophobicity of the protein (protein moieties), the stronger its interaction with the column or membrane. 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 sample containing the protein of interest.

[0477] Similar to ion exchange chromatography, HIC columns or HIC membrane devices can also be operated in elution, flow-through, or mixing modes, where the products exhibit binding or interaction with the chromatographic material and can be washed from such material using a buffer that is the same as or substantially similar to the loading buffer. (Details of these modes are outlined above regarding AEX processing.) In some embodiments, the HIC step is performed in negative mode, where process-related impurities bind to the immobilized ligands, and the protein of interest flows through.

[0478] Because hydrophobic interactions are strongest at high ionic strengths, this form of separation is best performed after a salt elution step (such as those typically used in conjunction with ion exchange chromatography). Alternatively, the salt can be added to the sample before the HIC step. High salt concentrations favor protein adsorption onto the HIC column, but the actual concentration can vary over a wide range depending on the nature of the protein of interest, the type of salt, and the specific HIC ligand chosen. Various ions can be configured in a so-called hydrophobic series depending on whether they promote hydrophobic interactions (salting out) or disrupt the water structure (liquid separation) and weaken the hydrophobic interactions. Cations are arranged in order of increasing salting out, starting with Ba. 2+ Ca 2+ Mg 2+ Li + Cs + Na + K + 、Rb + NH4 + Meanwhile, anions are gradually released into PO42- through the ionization process. 3- SO4 2- CH3CO3 - CI - ,Br - NO3 - ClO4 - I - SCN - .

[0479] HIC media typically contain an alkaline matrix (e.g., cross-linked agarose or synthetic copolymer materials) coupled to hydrophobic ligands (e.g., alkyl or aryl groups). Suitable HIC media contain agarose resins or phenyl-functionalized membranes (e.g., phenyl Sepharose from Stofahan). TM Alternatively, phenyl membranes from Sartorius. Various HIC resins are commercially available. Examples include, but are not limited to, Captophenyl, Captobutyl, and low- or high-substituted fast-flow phenyl Sepharose. TM 6. High-efficiency phenyl sepharose TM Highly effective octyl sepharoseTM (GE Healthcare); Fractogel TM EMDpropyl or Fractogel TM EMD phenyl (E.Merck, Germany); Macro-Prep TM Methyl or Macro-Prep TM Tert-butyl columnar (Bio-Rad, California); WP HI-propyl (C3) TM (JT Baker, New Jersey); and Toyopearl TM Ethers, 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 application-specific. However, such conditions can be advantageous because specific pH values ​​have minimal impact on the final selectivity and resolution of HIC separations in the pH 5.0–8.5 range. At pH values ​​above 8.5 or below 5.0, increasing pH weakens hydrophobic interactions and alters protein retention more significantly. Furthermore, changes in ionic strength, the presence of organic solvents, temperature, and pH (especially at the isoelectric point pI, when no net surface charge is present) can affect protein structure and solubility, and thus interactions with other hydrophobic surfaces (such as those in HIC culture media). Therefore, in some embodiments, this disclosure incorporates production strategies in which one or more of the aforementioned strategies are modulated to achieve a desired reduction in process-related impurities and / or product-related substances.

[0481] In some exemplary embodiments, spectroscopic methods such as UV, NIR, FTIR, fluorescence, and Raman spectroscopy can be used to monitor proteins and impurities of interest in linear, near-linear, or online modes, which can then be used to control the aggregate content in the collected material from the HIC adsorbent effluent. In other exemplary embodiments, linear, near-linear, or online monitoring methods can be used on the effluent line or in the collection container of the chromatographic step to enable the achievement of desired product quality / recovery. In other exemplary embodiments, UV signals can be used as an alternative 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 average, to address common process variations and achieve the target product quality. In some exemplary embodiments, such measurements can be combined with online dilution methods so that the ion concentration / conductivity of the loading / washing solution can be feedback-controlled and thus facilitate product quality control.

[0482] In some embodiments, the concentration of protein loaded on 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, etc. L, approximately 105 g / L, approximately 110 g / L, approximately 115 g / L, approximately 120 g / L, approximately 125 g / L, approximately 130 g / L, approximately 135 g / L, approximately 140 g / L, approximately 145 g / L, approximately 150 g / L, approximately 155 g / L, approximately 160 g / L, approximately 165 g / L, approximately 170 g / L, approximately 175 g / L, approximately 175 g / L, approximately 180 g / L, approximately 185 g / L, approximately 190 g / L, approximately 195 g / L, or approximately 200 g / L.

[0483] In some embodiments, the sample may be conditioned prior to contact with the HIC material; this can be referred to as loading conditioning. In some embodiments, a sample, such as a CEX aggregate, is conditioned to about 40 mM citrate using a single sling injection of about 1.2 M sodium citrate. After conditioning, the pH of the sample (HIC-loaded) 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 includes values ​​between approximately 14.00 ms / cm and approximately 17.00 ms / cm, approximately 14.00 ms / cm, approximately 14.10 ms / cm, approximately 14.20 ms / cm, approximately 14.30 ms / cm, approximately 14.40 ms / cm, approximately 14.50 ms / cm, approximately 14.60 ms / cm, approximately 14.70 ms / cm, approximately 14.80 ms / cm, approximately 14.90 ms / cm, approximately 15.00 ms / cm, approximately 15.10 ms / cm, approximately 15.20 ms / cm, and approximately 15.30 ms / cm. Conductivity of approximately 15.40 mS / cm, approximately 15.50 mS / cm, approximately 15.60 mS / cm, approximately 15.70 mS / cm, approximately 15.80 mS / cm, approximately 15.90 mS / cm, approximately 16.00 mS / cm, approximately 16.10 mS / cm, approximately 16.20 mS / cm, approximately 16.30 mS / cm, approximately 16.40 mS / cm, approximately 16.50 mS / cm, approximately 16.60 mS / cm, approximately 16.70 mS / cm, approximately 16.80 mS / cm, approximately 16.90 mS / cm, or approximately 17.00 mS / cm. In some embodiments, the concentration of citrate in the HIC loading is between about 10 mM and about 50 mM, between about 30 mM and about 40 mM, and is 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 undergoes a pre-equilibration step before contact with the sample. The pre-equilibration step removes all residual bound impurities from the column. In some embodiments, the pre-equilibration step is performed only for the first cycle of the batch. In some exemplary embodiments, the pre-equilibration buffer contains 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, and is 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 equilibrated before contact with the sample and optionally after a pre-equilibration step. The equilibration step alters the pH and conductivity of the mobile phase to match the buffer excipients present in the loaded material (sample). In some embodiments, the equilibration buffer may contain 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, and is 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 two column volumes of equilibration buffer are used.

[0486] In some embodiments, the protein concentration loaded in 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, and is about 10.0 g / L, about 10.5 g / L, about 11.0 g / L, about 11.5 g / L, about 12.0 g / L, about 12.5 g / L, about 13.0 g / L, about 13.5 g / L, about 14.0 g / L, about 14.5 g / L, about 15.0 g / L, about 15.5 g / L, about 16.0 g / L, about 16.5 g / L, about 17.0 g / L, about 17.5 g / L, about 18.0 g / L, about 18.5 g / L, about 19.0 g / L, about 19.5 g / L, or about 20.0 g / L. In some embodiments, the linear speed of the HIC loading is between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, and is 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 speed is about 150 cm / hr.

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

[0488] In some embodiments, the equilibration buffer is also used as a washing buffer. In some embodiments, the washing buffer contains about 40 mM Tris and about 40 mM sodium citrate, has a pH between about 6.30 and about 6.70, and has a conductivity between about 8.50 mS / cm and about 12.00 mS / cm. In some embodiments, the linear velocity of the washing buffer is between about 100 and about 300 cm / hr, between about 150 and about 250 cm / hr, and is 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 150 cm / hr. In some embodiments, the wash length is between about 6 and about 8 column volumes (CV), specifically about 6CV, about 6.5CV, about 7CV, about 7.5CV, or about 8CV. In one exemplary embodiment, the wash length is about 8CV.

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

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

[0491] Size exclusion chromatography

[0492] Size exclusion chromatography or gel filtration relies on the separation of components depending on their molecular size. Separation depends on the amount of time a substance spends in a porous stationary phase compared to its time in a fluid. The probability that a molecule will remain in the pores depends on the size of both the molecule and the pores. Furthermore, the ability of a substance to penetrate the pores is determined by the diffusion mobility of large molecules, which is higher for small macromolecules. Very large macromolecules cannot penetrate the pores of the stationary phase at all, and for very small macromolecules, the probability of penetration is close to 1 (unity). Components with larger molecular sizes move through the stationary phase more quickly, while components with smaller molecular sizes have a longer path through the pores of the stationary phase and therefore remain in the stationary phase for a longer time.

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

[0494] Porous chromatographic resins suitable for size exclusion chromatography of viruses can be made from dextrose, agarose, polyacrylamide, or silica with different physical properties. Polymer combinations can also be used. The most commonly used is the polymer combination "SEPHADEX," available from Amersham Biosciences. Other size exclusion supports from different constructing materials are also suitable, such as Toyopearl 55F (polymethacrylate, from Tosoh Bioscience, Montgomery Pa.) and Bio-Gel P-30Fine (BioRad Laboratories, Hercules, CA).

[0495] Virus interception and filtration

[0496] Virus filtration is a dedicated virus reduction step in the production process. This step is typically performed after chromatographic polishing. Virus reduction via a virus-retaining membrane is based on differences in hydrodynamic radii, 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) Asahi Kasei Pharmaceutical's Planova 20N. TM 50N or BioEx; EMD Millipore's Viresolve TM Filters; Sartorius CPV or HF; or Pall's Ultipor DV20 filter, DV50 TM Or a Pegasus Prime filter. Choosing the right filter to achieve the desired filtration performance is obvious to most technicians in the field.

[0498] The virus retention filtration step may additionally include a pre-filtration step. Virus retention filters are prone to premature fouling due to pore blockage caused by impurities present in the feed stream. Since the flux decline of related virus retention filters is associated with viral breakthrough in some virus retention filters, fouling is undesirable. Studies have reported using protective filters or pre-filters to improve virus filter fouling. The inventors have discovered that chemically functionalized pre-filters (including HIC, CEX, and AEX modes) improve virus retention filtration capacity. Pre-filtration can be achieved by using suitable pre-filters, including (but not limited to) Viresolve Shield, Viresolve Shield H, Millistak+HC Pro X0SP (MilliporeSigma) or Pall's Pegasus Protect. Those skilled in the art will appreciate that selecting a suitable pre-filter will achieve the desired filtration performance.

[0499] In some implementations, a virus interception filtering (VRF) step is performed immediately following the HIC step. In some implementations, the loading material is conditioned by three pre-filters that precede the three filters.

[0500] In some exemplary embodiments, the VRF process includes the following six steps. The filter assembly is first started and wetted with WFI until the unit is filled and properly ventilated at an operating transmembrane pressure (TMP) of 5 ± 2 psi. The second step includes using a flow rate greater than 50 L / m at an operating TMP of 25 ± 5 psi. 2 The solution is then rinsed with WFI. Following this, a pre-use integrity test is performed using sterile, oil-free air or nitrogen. The fourth step includes buffer rinsing and flux validation: using a flow rate greater than 20 L / m³ at 25 ± 5 psi TMP. 2 The volume was equilibrated with a pH 6.5 buffer containing approximately 40 mM Tris and approximately 40 mM sodium citrate.

[0501] Subsequently, at a constant 25±5 psi TMP, with a concentration of less than 900 L / m 2 The sample, such as a HIC aggregate, is loaded in a specific volume. Aggregate collection begins immediately after sample loading and ends once the loaded material is exhausted. In some embodiments, the sample volume is approximately 700 L / m³. 2 With approximately 1300L / m 2 Between, approximately 700 L / m 2 Approximately 750 L / m 2 Approximately 800L / m 2 Approximately 850 L / m 2 Approximately 900 L / m 2 Approximately 950 L / m 2 Approximately 1000 L / m 2 Approximately 1050 L / m 2 Approximately 1100L / m 2 Approximately 1150 L / m 2 Approximately 1200L / m 2 Approximately 1250 L / m 2 Approximately 1300L / m 2 Approximately 1350 L / m 2 Approximately 1400 L / m 2 Approximately 1500 L / m 2 Approximately 1600 L / m 2 Approximately 1700 L / m 2 Approximately 1800 L / m 2 Approximately 1900 L / m 2 Or approximately 2000 L / m 2In some embodiments, the conductivity is loaded between about 7.5 mS / cm and about 13.5 mS / cm, specifically 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 pH is loaded between about 4.5 and about 7.5, between about 6.3 and about 6.7, specifically 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 protein concentration is between about 4.5 g / L and about 13 g / L, between about 8 g / L and about 10 g / L, and is 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 to prepare for a final post-use integrity test using sterile, oil-free air or nitrogen.

[0503] Concentration and Percolation

[0504] Some exemplary embodiments of this disclosure employ ultrafiltration and diafiltration (UF / DF), also known as concentration and diafiltration, to further concentrate and reconstitute the protein of interest. UF / DF adjusts the drug substance to achieve pH, excipient content, and protein concentration favorable for long-term storage and for the addition of stabilizing excipients to produce the final drug substance (FDS). During processing, the protein solution is tangentially pumped across the surface of a semi-permeable parallel sheet membrane. The membrane may have a pore size of, for example, about 50 kDa, which is permeable to water and buffer salts, but is generally impermeable to the protein of interest (e.g., a monoclonal antibody). The driving force for permeation is the applied transmembrane pressure (TMP) caused by flow restriction at the outlet of the membrane flow channel.

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

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

[0507] During the percolation phase, permeable solutes are replaced as fresh buffer is flushed into the product stream. The total system volume is defined by the sum of the osmotic tank and carriage volumes when fresh buffer is added at the same rate while removing permeate from the system. A turn-over volume (TOV) is defined as the amount of percolation buffer added to the UF / DF process to match the system volume. Typically, replacing 8 times the system volume (8 TOV) ensures >99.9% buffer exchange. The percolation buffer is designed to adjust proteins to a stable pH and compensate for high product concentrations with excipient concentrations.

[0508] Once sufficient buffer exchange has occurred, secondary concentration further reduces the product volume to facilitate storage and reconstitution. If further reduction in product volume is required, for example due to equipment assembly considerations, higher final protein concentration requirements, or the need for viscosity modifier adjustments, then final concentration is performed.

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

[0510] The exemplary steps are described again in more detail. The first UF / DF step includes WFI flushing, with a residual volume of approximately 12 L / m³. 2 And the permeate is 40 L / m 2 The feed flow rate is approximately 200 to 300 L / hr / m. 2( The pressure was maintained between LMH and approximately 25 ± 5 psi. Next, a pre-use leak test was performed using WFI at an inlet pressure of 8–12 psi. Equilibration was then carried out with approximately 4 mM acetate at approximately pH 4.10 ± 0.10. The equilibration buffer volume was greater than or approximately 5 L / m³. 2 And the flow rate is between approximately 200 and 300 LMH.

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

[0512] Next, an initial concentration step is performed using a loading agent, such as a VRF aggregate, to achieve a concentration of approximately or less than 970 g / m³. 2 The volumetric loading is increased at a flow rate between approximately 200 and 350 LMH, and at a pressure of approximately 20 ± 10 psi. In one exemplary embodiment, the flow rate is approximately 300 LMH. The initial concentrated protein concentration can be between approximately 60 and 80 g / L. In one exemplary embodiment, the protein concentration at this step is approximately 70 g / L.

[0513] The initial concentration step is followed by a percolation step. The percolation buffer may contain about 4 mM acetate with a pH between about 4.00 and 4.20. In some embodiments, the acetate concentration in the percolation buffer is between about 3 mM and about 10 mM, between about 3 mM and about 5 mM, or 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 percolation buffer is between about 4.00 and about 4.50, specifically 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 one exemplary embodiment, the pH of the percolation buffer is about 4.10.

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

[0515] Following percolation is a secondary concentration step. The amount of product used for secondary concentration is approximately or less than 970 g / m³. 2 The flow rate for secondary concentration is between approximately 100 and approximately 350 LMH. In one exemplary embodiment, the flow rate is approximately 300 LMH. The pressure for secondary concentration is between approximately 10 and 30 psi, with an inlet pressure of less than 60 psi. The protein concentration after secondary concentration can be between approximately 80 and 100 g / L. In one exemplary embodiment, the protein concentration after secondary concentration is approximately 90 g / L.

[0516] Following the secondary concentration is the membrane depolarization step. This process can last between approximately 5 and 15 minutes, with a flow rate of approximately or less than 350 LMH and a pressure less than 30 psi. For this step, the permeate valve is 0% open, while the residue valve is 100% open.

[0517] The next step is the final or third concentration step. The final concentrate used may be loaded at a rate of approximately or less than 1800 g / m³. 2The protein is added at a flow rate between approximately 5 and 350 LMH and an inlet pressure of less than 60 psi. In some embodiments, the final protein concentration is between approximately 180 g / L and approximately 260 g / L, between approximately 225 g / L and approximately 255 g / L, between approximately 228 g / L and approximately 232 g / L, and is approximately 180 g / L, approximately 185 g / L, approximately 190 g / L, approximately 195 g / L, approximately 200 g / L, approximately 205 g / L, approximately 210 g / L, approximately 215 g / L, approximately 220 g / L, approximately 225 g / L, approximately 230 g / L, approximately 235 g / L, approximately 240 g / L, approximately 245 g / L, approximately 250 g / L, approximately 255 g / L, or approximately 260 g / L. In one exemplary embodiment, the final protein concentration is approximately 232 g / L. In another exemplary embodiment, the final protein concentration is approximately 240 g / L.

[0518] Following final concentration, the method includes an additional membrane depolarization step lasting approximately 15 to 35 minutes. In one exemplary embodiment, this step lasts approximately 25 minutes. The feed flow rate is approximately or less than 350 LMH, the pressure is below 30 psi, the permeate valve is 0% open, and the residue valve is 100% open.

[0519] Finally, after membrane polarization, a WFI rinsing step is performed, with a flow rate of 10 L / m applied at a rate between approximately 200 and approximately 250 LMH. 2 WFI.

[0520] preparation

[0521] Some exemplary embodiments further include adjusting the final concentrated aggregate (FCP) protein after the concentration and percolation steps. The concentrated protein can be adjusted to the desired concentration with the desired excipients for use in the final drug substance (FDS).

[0522] If the concentration of the filtered FCP protein is higher than the target concentration, it is diluted with FCP dilution buffer to adjust it to the target concentration. The FCP dilution buffer may contain approximately 10 mM sodium acetate, approximately 25 mM arginine hydrochloride, and approximately 5% sucrose, with a pH of approximately 5.3. In some embodiments, the concentration of sodium acetate in the FCP dilution buffer may be between approximately 2.0 mM and approximately 14.0 mM, between approximately 6.0 mM and approximately 12.0 mM, between approximately 8.0 mM and approximately 10.0 mM, approximately 2.0 mM, approximately 2.5 mM, approximately 3.0 mM, approximately 3.5 mM, approximately 4.0 mM, approximately 4.5 mM, approximately 5 mM, and approximately 5.5 mM. mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11.0 mM, about 11.5 mM, about 12.0 mM, about 12.5 mM, about 13.0 mM, about 13.5 mM, or about 14.0 mM. In some embodiments, the concentration of arginine hydrochloride in the FCP dilution buffer may be between about 10 mM and about 40 mM, between about 15 mM and about 35 mM, between about 20 mM and about 30 mM, or 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 may be between about 1% and 10%, between about 2% and 9%, between about 3% and 8%, or 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 may be between about 4.0 and about 6.5, between about 4.5 and about 6.0, between about 5.0 and about 5.5, or 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 may 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 may contain about 10 mM acetate, about 350 mM arginine hydrochloride, and about 70% (w / v) sucrose, with a pH of about 5.3. In some embodiments, the acetate concentration in the excipient concentration 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, or 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, or about 5.5 mM. mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11.0 mM, about 11.5 mM, about 12.0 mM, about 12.5 mM, about 13.0 mM, about 13.5 mM, or about 14.0 mM. In some embodiments, the concentration of arginine hydrochloride in the excipient concentrate buffer may be between about 200 mM and about 500 mM, between about 250 mM and about 450 mM, between about 300 mM and about 400 mM, or about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM. In some embodiments, the concentration of sucrose in the excipient concentrate buffer may be between 50% and 90%, between about 55% and 85%, between about 60% and 80%, or 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 concentrate buffer may be between about 4.0 and about 6.5, between about 4.5 and about 6.0, between about 5.0 and about 5.5, or 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 concentrate buffer, the regulated FCP can be filtered into a single-use bag and mixed. The sample can be referred to as the filtered active pharmaceutical ingredient (DS). The filtered DS can be further diluted to the target concentration with FDS dilution buffer and mixed with excipient buffer solution to form the final concentration and composition.

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

[0526] The FDS dilution buffer may contain approximately 10 mM sodium acetate, approximately 25 mM arginine hydrochloride, and approximately 5% sucrose, with a pH of approximately 5.3. In some embodiments, the concentration of sodium acetate in the FDS dilution buffer may be between approximately 2.0 mM and approximately 14.0 mM, between approximately 6.0 mM and approximately 12.0 mM, between approximately 8.0 mM and approximately 10.0 mM, approximately 2.0 mM, approximately 2.5 mM, approximately 3.0 mM, approximately 3.5 mM, approximately 4.0 mM, approximately 4.5 mM, approximately 5 mM, and approximately 5.5 mM. mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 9.5 mM, about 10.0 mM, about 10.5 mM, about 11.0 mM, about 11.5 mM, about 12.0 mM, about 12.5 mM, about 13.0 mM, about 13.5 mM, or about 14.0 mM. In some embodiments, the concentration of arginine hydrochloride in the FDS dilution buffer may be between about 10 mM and about 40 mM, between about 15 mM and about 35 mM, between about 20 mM and about 30 mM, or about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, or about 40 mM. In some embodiments, the concentration of sucrose in the FDS dilution buffer may be between about 1% and 10%, between about 2% and 9%, between about 3% and 8%, or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the pH of the FDS dilution buffer may be between about 4.0 and about 6.5, between about 4.5 and about 6.0, between about 5.0 and about 5.5, or 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 based on the target concentration and composition of the final drug substance (FDS).

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

[0529] The excipient buffer solution with a target FDS concentration of 175 mg / mL may 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 (target FDS concentration of 175 mg / mL) may be between about 15 mM and about 45 mM, between about 20 mM and about 40 mM, between about 25 mM and about 35 mM, or 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) may 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, or 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) may 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, or 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) may be between about 1% and 10%, between about 2% and 9%, between about 3% and 8%, or 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) may 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%, or 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) may 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, or 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] Combined assessment of total insulin concentration (0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L) and insulin schedule (all insulin added on day 4; insulin added equally on days 2 and 4; insulin added equally on days 2, 4, 6, and 8).

[0533] A modified seed expansion culture was conducted using normal operating conditions in flasks and bioreactors, with inoculation tailored to a D-optimal design as shown in Table 9 below. A 15-day batch feeding process was carried out in a 2L bioreactor containing CDM1B containing taurine and sodium phosphate (Table 10).

[0534] Table 9 (Experimental Conditions for Insulin Supplementation)

[0535]

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

[0537]

[0538] Cell counts and metabolites were measured in bioreactor samples using a Nova Flex analyzer. Samples were analyzed for titer analysis after production was completed. When the production duration was 15 days, bioreactor samples from day 13 were analyzed for product quality analysis.

[0539] In some bioreactors, increased insulin leads to higher productivity. Figure 2 ), higher survival rate Figure 3 ) and overall reduction in ammonia ( Figure 4 Higher inoculation densities also affect titer profile parameters and cause a decrease in ammonia and reduced survival rates.

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

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

[0542] Example 2. Tyrosine supplementation

[0543] It should be noted that several amino acids are depleted during the process of increasing productivity. Therefore, additional tyrosine feed should be investigated to address the depletion problem.

[0544] A modified seed culture was conducted in flasks under normal operating conditions, following a D-optimal design for the inoculation flasks as shown in Table 11. A 15-day batch feeding process was performed in 250 mL flasks containing cell culture medium containing sodium phosphate and taurine. Tyrosine was added on day 3 of production. The tyrosine concentration was varied between 1.8 g / L and 2.2 g / L according to the experimental design (Table 11).

[0545] Table 11

[0546]

[0547] Feeding extra tyrosine into cell production causes a decrease in ammonia and has a negligible effect on productivity, such as Figure 9 As shown in the image.

[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 unwanted cellular metabolic byproducts. Initial small-scale model development experiments indicated that phosphate may be depleted by day 2. Therefore, phosphate redistribution, including increasing the concentration of the basal medium, was investigated.

[0550] Evaluate phosphate feed arrangements in a production flask model. Avoiding depletion of key nutrients, including phosphate, is fundamental and essential before initiating more complex culture medium and feed studies.

[0551] Production flask experiments were conducted using a modified seed culture for expansion, inoculated into flasks and seed culture bioreactors. Batch feeding of selected cell lines was carried out in flasks containing a starting production medium containing taurine. The sodium phosphate content in the starting medium was varied according to the experimental design. The sodium phosphate content also varied in the four feeds throughout the production process (n=2 for each condition, see Table 12).

[0552] Table 12

[0553]

[0554]

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

[0556] Cell counts of daily samples were analyzed on a Nova CDV, metabolites of daily samples were analyzed on a Nova BP400, and osmolar concentrations of daily samples were analyzed on an Advanced Instruments Model 2020 osmometer. Sample titer analysis was also provided.

[0557] The phosphate redistribution strategy resulted in a slight increase in the final titer. Figure 10 Different feed paradigms alter the shape of the phosphate concentration curve, and three conditions were observed where phosphate was nearly exhausted at different production days. Figure 11 The conditions of adding phosphate early in the batch and continuously feeding phosphate until the end of the batch (267, 525, 525, 250, 250 mg / L) did not deplete the phosphate during production; this feeding strategy is implemented for upstream processes.

[0558] Example 4. Production Bioreactor Conditions

[0559] The cell culture parameters in the expected production bioreactor have the greatest impact on product quality and process productivity, as most proteins are produced in this unit operation.

[0560] Two N-3 10L stirred tank bioreactors were inoculated with culture from the seed expansion culture. After reaching the final N-3 density, three N-2 10L stirred tank bioreactors were inoculated. After reaching the final N-2 density, four N-1 10L stirred tank bioreactors were inoculated. An appropriate split ratio was used throughout the seed expansion culture. At three specified final N-1 density targets, 2L batch-fed production bioreactors were inoculated. Three of the four N-1 reactors were used to inoculate the production bioreactors. The lower pH limit was kept constant except for inoculation-related variables such as density, temperature, and upper pH limits, which were set according to the experimental design.

[0561] Cell counts and metabolites were analyzed from daily samples on Nova FLEX. After production batch completion, samples from days 10, 12, and / or 13 were analyzed for titer analysis and quality testing. Sequence variants from sixteen samples from days 10, 12, and / or 13 were also analyzed.

[0562] It has been determined that the collection date has a significant impact on initial general quality properties, process properties, and sequence variants (titer, total fucosylation, total galactosylation, viability, total Pro-Ala SV, Pro-Ala site P12). The collection date is a time-dependent upstream process parameter, where the operating range is defined considering production equipment capacity and flexibility. Monte Carlo simulations are performed to determine combinations of ranges. An optimal profiler based on the JMP intention-maximization algorithm determines day 10.0 as the optimal setpoint.

[0563] Monte Carlo simulations were performed on the N-1 density to characterize the process failure rate and quality properties of the combined test process range. The ideal process range was then selected based on achieving the most desirable manufacturing process, with a focus on minimizing the failure rate (target failure rate <100 ppm, 0.01%). Fail...

Claims

1. A method comprising the following steps: (a) subjecting the collected Dupilumab to affinity chromatography; (b) subjecting the pooled dupilumab from the eluate of step (a) to viral inactivation at a pH of about 3 to about 4 and then adjusting the pH to about 5 to about 8; (c) subjecting the Dupilumab pooled from step (b) to anion exchange chromatography in flow-through mode; (d) subjecting the dupilumab pooled from the flow-through fractions of step (c) to cation exchange chromatography in a bind and elute mode; (e) subjecting the pooled dupilumab from the eluate of step (d) to hydrophobic interaction chromatography in flow-through mode; and (f) subjecting the dupilumab pooled from the flow-through fractions of step (e) to virus-retaining filtration to produce dupilumab.

2. The method according to claim 1, further comprising a collection pretreatment step before step (a).

3. The method of claim 2, wherein the collection pretreatment step comprises adjusting the dupilumab to an instantaneous pH level of about 4 to 5.

5.

4. The method of claim 1, wherein the concentration of the dupilumab pooled from the flow-through fractions in step (f) is between about 4 g / L and 12 g / L.

5. The method of claim 1, further comprising subjecting the dupilumab to ultrafiltration and diafiltration (UF / DF) after step (f).

6. The method of claim 5, wherein the UF / DF comprises a diafiltration buffer having a pH between 4.0 and 4.

5.

7. The method of claim 5, wherein the pH of the concentrated depilumab pool after UF / DF is about 5.

3.

8. The method of claim 5, wherein the UF / DF comprises a diafiltration buffer comprising about 4 mM acetate to about 6 mM acetate.

9. The method of claim 1, wherein the affinity chromatography is Protein A chromatography.

10. The method of claim 9, wherein the Protein A resin is selected from the group consisting of MabSelect PrismA, MabSelect SuRe, MabSelect SuRe LX, MabSelect, MabSelect SuRe pcc, MabSelect Xtra, rProtein A Sepharose, ProSep HC, ProSep Ultra, ProSep Ultra Plus, MabCapture, and Amsphere A3.

11. The method of claim 9, wherein the Protein A resin is selected to be able to accept a loading concentration greater than 55 g / L.

12. The method of claim 1, wherein about 180 g to 200 g of dupilumab is loaded per liter of HIC resin.

13. The method of claim 1, wherein the amount of PLBD2 in the HIC eluate is reduced compared to the amount of PLBD2 in the HIC load, optionally wherein the amount of PLBD2 in the HIC eluate is reduced to less than 100 ppm, to less than 30 ppm, to less than 4 ppm, to less than 1 ppm, or by about 40× to 310× compared to the amount of PLBD2 in the HIC load. The method of claim 9 , wherein the loading pH of the Protein A column is between 6 and 8.

15. The method of claim 1, wherein the dupilumab 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.

16. The method of claim 1, wherein the dupilumab 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.

17. A method comprising the steps of: (a) culturing cells expressing dupilumab; (b) subjecting the cells to an instantaneous pH level of about 4.0 to 5.5, followed by raising the pH level to about 5.5 to 6.5; (c) collecting the cells by centrifugation to separate cell debris from the clarified culture medium containing the dupilumab; (d) subjecting the clarified medium to affinity chromatography; (e) subjecting the pooled dupilumab from the eluate of step (d) to viral inactivation at a pH of about 3 to about 4 and then adjusting the pH to about 5 to about 8; (f) subjecting the pooled dupilumab from step (e) to anion exchange chromatography in flow-through mode; (g) subjecting the dupilumab pooled from the flow-through fractions of step (f) to cation exchange chromatography in a bind and elute mode; (h) subjecting the pooled dupilumab from the eluate of step (g) to hydrophobic interaction chromatography in flow-through mode; and (i) subjecting the dupilumab pooled from the flow-through fractions of step (h) to virus-retaining filtration to produce dupilumab.

18. The method of claim 17, further comprising subjecting the dupilumab to ultrafiltration and diafiltration (UF / DF) after step (i).

19. The method of claim 17, wherein the affinity chromatography is Protein A chromatography.

20. The method of claim 19, wherein the Protein A resin is selected from the group consisting of MabSelect PrismA, MabSelect SuRe, MabSelect SuRe LX, MabSelect, MabSelect SuRe pcc, MabSelect Xtra, rProtein A Sepharose, ProSep HC, ProSep Ultra, ProSep Ultra Plus, MabCapture, and AmsphereA3.

21. The method of claim 17, wherein the anion exchange resin is selected from the group consisting of Q Sepharose Fast Flow, Poros 50PI, Poros 50HQ, Capto Q Impres, Capto DEAE, Toyopearl QAE-550, Toyopearl DEAE-650, Toyopearl GigaCap Q-650, Fractogel EMDTMAE Hicap, Sartobind STIC PA nano, Sartobind Q nano, CUNO BioCap, and XOHC.

22. The method of claim 17, wherein the cation exchange resin is selected from the group consisting of Fractogel Hicap, Capto SPImpRes, Capto S ImpAc, Grade F CM Hyper D, Eshmuno S, Nuvia C Prime, Nuvia S, Poros HS and Poros XS.

23. The method of claim 17, further comprising passing the dupilumab through a LifeAssure filter after the viral inactivation of step (e) and before the anion exchange chromatography of step (f).

24. The method of claim 18, wherein the UF / DF step comprises a membrane filtration device selected from the group consisting of Pellicon 2, Pellicon 3 filtration cassettes having 10 kD, 30 kD or 50 kD membranes, Kvick 10 kD, 30 kD or 50 kD membrane filtration cassettes, and Centramate and Centrasette 10 kD, 30 kD or 50 kD filtration cassettes.

25. The method of claim 18, wherein the UF / DF step does not include the addition of arginine.

26. The method of claim 17, wherein the HIC step comprises a HIC medium selected from the group consisting of Capto Phenyl, High Substituted Capto Phenyl, Fast Flow Phenyl Sepharose TM 6. High-efficiency phenyl Sepharose TM , Octyl Sepharose High Performance, Fractogel EMD Propyl, Fractogel EMD Phenyl, Macro-Prep Methyl, Macro-Prep tert-Butyl, WP HI-Prop (C3), Toyopearl Ether, Phenyl or Butyl, ToyoPPG, Toyo Phenyl, Toyo Butyl and Toyo Hexyl.

27. The method of claim 17, wherein the dupilumab 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.

28. The method of claim 17, wherein the dupilumab 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.

29. A method for producing an anti-IL4Rα antibody or an antigen-binding fragment thereof, comprising: (a) subjecting the collected antibodies or antigen-binding fragments thereof to affinity chromatography; (b) subjecting the antibodies or antigen-binding fragments thereof pooled from the eluate of step (a) to viral inactivation; (c) subjecting the antibodies or antigen-binding fragments thereof pooled from step (b) to anion exchange chromatography in flow-through mode; (d) subjecting the antibodies or antigen-binding fragments thereof pooled from the flow-through fractions of step (c) to cation exchange chromatography in a bind and elute mode; (e) subjecting the antibodies or antigen-binding fragments thereof pooled from the eluate of step (d) to hydrophobic interaction chromatography in flow-through mode; (f) subjecting the antibodies or antigen-binding fragments thereof pooled from the flow-through fraction of step (e) to virus-retaining filtration; and (g) subjecting the antibodies or antigen-binding fragments thereof pooled from step (f) to ultrafiltration and diafiltration (UF / DF) to produce an anti-IL4Rα antibody or antigen-binding fragment thereof, wherein the UF / DF step does not include the addition of arginine.

30. The method of claim 29, wherein the diafiltration buffer of step (g) has a pH between 4.0 and 4.

5.

31. The method of claim 30, wherein the diafiltration buffer comprises about 4 mM acetate to about 6 mM acetate.

32. The method of claim 29, wherein the pH of the concentrated pool after UF / DF is about 5.

3.

33. The method of claim 29, wherein the UF / DF step comprises a membrane filtration device selected from the group consisting of Pellicon 2, Pellicon 3 filtration cassettes having 10 kD, 30 kD or 50 kD membranes, Kvick 10 kD, 30 kD or 50 kD membrane filtration cassettes, and Centramate and Centrasette 10 kD, 30 kD or 50 kD filtration cassettes.

34. A method comprising the steps of: (a) subjecting the collected antibodies to affinity chromatography; (b) subjecting the pooled antibodies from the eluate of step (a) to viral inactivation at a pH of about 3 to about 4 and subsequently adjusting the pH to about 5 to about 8; (c) subjecting the antibodies pooled from step (b) to anion exchange chromatography in flow-through mode; (d) subjecting the antibodies pooled from the flow-through fractions of step (c) to cation exchange chromatography in a bind and elute mode; (e) subjecting the antibodies pooled from the eluate of step (d) to hydrophobic interaction chromatography in flow-through mode; and (f) subjecting the antibodies pooled from the flow-through fraction of step (e) to virus-retaining filtration to produce anti-IL4Rα antibodies.

35. The method of claim 34, further comprising a collection pretreatment step prior to step (a).

36. The method of claim 35, wherein the collection pretreatment step comprises adjusting the antibody to an instantaneous pH level of about 4 to 5.

5.

37. The method of claim 34, wherein the concentration of the antibody pooled from the flow-through fraction in step (f) is between about 4 g / L and 12 g / L.

38. The method of claim 34, further comprising subjecting the antibody to ultrafiltration and diafiltration (UF / DF) after step (f).

39. The method of claim 38, wherein the UF / DF comprises a diafiltration buffer having a pH between 4.0 and 4.

5.

40. The method of claim 38, wherein the pH of the concentrated antibody pool after UF / DF is about 5.

3.

41. The method of claim 38, wherein the UF / DF comprises a diafiltration buffer comprising about 4 mM acetate to about 6 mM acetate.

42. The method of claim 34, wherein the affinity chromatography is Protein A chromatography.

43. The method of claim 42, wherein the Protein A resin is selected from the group consisting of MabSelect PrismA, MabSelect SuRe, MabSelect SuRe LX, MabSelect, MabSelect SuRe pcc, MabSelect Xtra, rProtein A Sepharose, ProSep HC, ProSep Ultra, ProSep Ultra Plus, MabCapture, and Amsphere A3.

44. The method of claim 42, wherein the Protein A resin is selected to be capable of accepting a loading concentration greater than 55 g / L.

45. The method of claim 34, wherein about 180 g to 200 g of antibody is loaded per liter of HIC resin.

46. ​​The method of claim 34, wherein the amount of PLBD2 in the HIC eluate is reduced compared to the amount of PLBD2 in the HIC load, optionally wherein the amount of PLBD2 in the HIC eluate is reduced to less than 100 ppm, reduced to less than 30 ppm, reduced to less than 4 ppm, reduced to less than 1 ppm, or reduced by about 40× to 310× compared to the amount of PLBD2 in the HIC load.

47. The method of claim 42, wherein the loading pH of the Protein A column is between 6 and 8.

48. The method of claim 34, wherein 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.

49. The method of claim 34, wherein 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.

50. The method of claim 34, wherein the anti-IL-4Rα antibody is dupilumab.

51. A method for treating a patient suffering from a type 2 inflammatory disease, comprising administering to the patient an anti-IL4Rα antibody produced according to the method of any one of claims 1 to 50.

52. The method of claim 51, 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.

53. A method for treating a disease or disorder associated with IL-4R activity, comprising administering to a patient an anti-IL4Rα antibody produced according to the method of any one of claims 1 to 50.

54. The method of claim 53, wherein the disease or condition associated with IL-4R activity is atopic dermatitis, moderate to severe atopic dermatitis, asthma, moderate to severe asthma, allergic rhinitis, chronic sinusitis with nasal polyps, eosinophilic esophagitis, chronic obstructive pulmonary disease, chronic spontaneous urticaria, prurigo nodularis, allergic fungal sinusitis, chronic sinusitis without nasal polyps, allergies, grass allergies, peanut allergies, dairy allergies, bullous pemphigoid, atopic dermatitis of the hands and feet, cold urticaria, chronic induced urticaria, ulcerative colitis, chronic pruritus of unknown origin, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, bronchiectasis, or alopecia areata.

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