Methods for modulating afucosylation of antibody products

Abstract

The present application is directed to a method for modulating afucosylation of an antibody product produced in a bioreactor, the method comprising adding mannose to the bioreactor to control afucosylation of the antibody product, including increasing afucosylation by 1% or more compared to an untreated bioreactor product.

Description

[Technical Field]

[0001] The present application is directed to a method for modulating afucosylation of an antibody product produced in a bioreactor, the method comprising adding mannose to the bioreactor to control afucosylation of the antibody product, including increasing afucosylation by 1% or more compared to the untreated bioreactor product. [Background technology]

[0002] Protein products, such as antibodies, undergo post-translational modifications during their expression from cells, including the attachment of sugar moieties. One such modification is the N-linked glycosylation of immunoglobulin G (IgG), which occurs at Asn 297 in the CH2 domain of the mammalian IgG heavy chain. This particular N-linked glycosylation is achieved by the initial addition of preformed oligosaccharides, which are then subjected to subsequent modifications that remove glucose and mannose residues and add other sugars, such as fucose, galactose, sialic acid, and N-acetylglucosamine (GlcNAc). Such glycosylation can have a profound effect on the biological activity of proteins. Specifically, antibody-dependent cellular cytotoxicity (ADCC), an important mechanism of action for many therapeutic antibodies, depends on the fucosylation level of the antibody. Various reports have found that monoclonal antibodies with reduced amounts of fucosylation (i.e., higher afucosylation) exhibit higher ADCC compared to their fucosylated counterparts. Thus, the production of antibody products that can have increased afucosylation is advantageous for several therapeutic approaches, particularly in oncology.

[0003] Furthermore, because the type and degree of glycosylation can affect biological activity, the glycan profile of a therapeutic antibody is an important critical quality attribute that must be reported to regulatory authorities and consistently reproduced. However, when the production of a therapeutic antibody is transferred from one process to another (or even between manufacturing sites), variations in CQAs such as fucosylation can occur, resulting in the need to adjust the transferred process to achieve fucosylation levels in the range previously achieved for that product.

[0004] Thus, there is a need to be able to control the level of fucosylation in recombinantly expressed antibody compositions to enhance biological activity and / or to match the fucosylation level to previous manufacturing processes. Summary of the Invention

[0005] Surprisingly, it has been found that adding mannose in amounts greater than about 1 g / L to an antibody production process results in a statistically significant increase in afucosylation of the antibody product (when compared to the same production process without the addition of mannose). Furthermore, adding mannose in such amounts in the present method does not appear to result in an increase in high-mannose species. These results are surprising and unexpected, and as described herein, the present method provides (i) a means to modulate the afucosylation of antibody products to produce therapeutic antibodies that can enhance ADCC and thus provide enhanced therapeutic efficacy, and (ii) a means to adjust afucosylation levels to ensure a consistent level, given that this is a critical quality attribute. The present method has significant advantages over other methods that require genetically engineered cell lines to enhance afucosylation (due to the difficulties in producing and maintaining such cell lines) and other methods that require significant manipulation of upstream bioprocessing parameters.

[0006] Thus, in one embodiment, the invention provides a method for increasing afucosylation of a recombinantly expressed antibody in a bioreactor, the method comprising culturing cells expressing the antibody in a bioreactor and adding mannose to the cell culture during the antibody production process, such that afucosylation of the antibody is increased compared to the same antibody produced by the antibody production process without the addition of mannose.

[0007] Also provided herein is a method for matching the afucosylation of a recombinantly produced antibody to a target afucosylation percentage previously obtained for the same antibody, the method comprising culturing cells expressing the antibody in a bioreactor and controlling the addition of mannose to the bioreactor during the antibody production process to obtain an expressed antibody with the target afucosylation percentage.

[0008] Other features and aspects of the disclosure are discussed in more detail below. DETAILED DESCRIPTION OF THE INVENTION

[0009] It should be understood by those skilled in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present disclosure.

[0010] In embodiments, provided herein are methods for modulating the afucosylation of antibody products, typically intended to (i) modify the biological activity of the antibody, e.g., increase antibody-dependent cellular cytotoxicity (ADCC), or (ii) fine-tune afucosylation in the process to match previously obtained levels.

[0011] "Fucosylation," a type of glycosylation, is the process of adding fucose sugar units to molecules, including proteins such as antibodies (also referred to herein as "antibody products"). As used herein, "afucosylation" refers to the absence of fucose sugar units on a particular molecule, such as a particular antibody product. In an antibody product preparation, the afucosylation level is the percentage of antibody molecules lacking fucose sugar units. This can be determined by a number of methods, including mass spectrometry and high-pressure liquid chromatography (HPLC). Because there may be some variation between the methods used, in one embodiment, the percentage is measured using a time-of-flight liquid chromatography / mass spectrometer (TOF LC / MS) system. The antibody is reduced and then loaded directly into a liquid chromatography / mass spectrometer (LC / MS), such as an Agilent 6230B TOF LC / MS system (no PNGase F digestion required), and the reduced antibody is passed through a reverse-phase desalting column on the liquid chromatograph before being injected into the time-of-flight mass spectrometer.

[0012] Other suitable methods include those described, for example, in Tay and Butler, 2015, J. Biol. Methods 2:19; Mishra et al., 2020, J. Biotechnology X 5:100015. In one of these described methods, glycans can be removed using PNGase F and dried. They are then labeled using 2-AB labels and analyzed using hydrophilic interaction liquid chromatography-HPLC (HILIC-HPLC).

[0013] The detected species are then analyzed to determine the percentage of afucosylated antibodies. Measurements are usually replicated to improve measurement accuracy.

[0014] In one embodiment, the fucosylation percentage is calculated based only on N-linked glycan species, such as those attached to the Fc domain of an antibody. N-linked glycan species include GO, GOF, GO ...

[0015] As used herein, the terms "antibody product" and "antibody" are used interchangeably, and an antibody product is the result of an antibody production process. As used herein, the terms "antibody" and "immunoglobulin" can be used interchangeably and refer to a polypeptide or group of polypeptides containing at least one binding domain formed from the folding of a polypeptide chain, which has a three-dimensional binding space with an internal shape and charge distribution complementary to the antigenic determinant characteristics of an antigen. Antibodies typically have a tetrameric form, with two pairs of polypeptide chains, each pair having one "light" chain and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by multiple constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain, and the light chain variable domain aligned with the variable domain of the heavy chain. Light chains are classified as either lambda or kappa chains based on the amino acid sequence of the light chain constant region.

[0016] Immunoglobulin molecules can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), subisotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or allotype (e.g., Gm, e.g., G1m(f, z, a, or x), G2m(n), G3m(g, b, or c), Am, Em, and Km(1, 2, or 3)). Immunoglobulins include, but are not limited to, monoclonal antibodies (mAbs) (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope-binding fragments (e.g., bispecific antibodies), CDR-grafted, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, anti-idiotypic (anti-Id) antibodies, intrabodies, and any desired antigen-binding fragment thereof, including recombinantly produced antibody fragments. Examples of antibody fragments that can be recombinantly produced include, but are not limited to, antibody fragments comprising variable heavy and light chain domains, such as single chain Fvs (scFvs), single chain antibodies, Fab fragments, Fab' fragments, and F(ab')2 fragments. Antibody fragments can also include epitope-binding fragments or derivatives of any of the antibodies listed above. In preferred embodiments, the antibody product is a monoclonal antibody (mAb), and more preferably, a therapeutic antibody product.

[0017] As described herein, antibody production processes are preferably carried out in bioreactors, which are vessels suitable for culturing producer cells expressing the antibody of interest. Because bioreactors are typically used at production scale, or pilot scale, before being scaled up for production, bioreactors used in production processes typically have a volume of at least 10 L; however, smaller bioreactors, such as the AMBR® 250 system, having a volume of 100 to 250 mL, can be used to test the process. Thus, in an exemplary embodiment, the bioreactor can have a volume of about 100 mL to about 50,000 L. Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, and 550 liters. Reactors may have volumes of 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. Suitable reactors may be multi-use, single-use, disposable, or non-disposable and may be formed from any suitable material, including stainless steel (e.g., 316L or any other suitable stainless steel) and metal alloys such as Inconel, plastic, and / or glass.

[0018] The antibody production process includes a cell culture or cell population producing the antibody product, along with a production medium or buffer that suitably contains the necessary reagents and supplements, including a suitable nutrient medium, to support cell growth and production of the desired antibody.

[0019] The total bioreactor fill volume, also referred to herein as the "production process volume," refers to the actual volume of cell culture in the bioreactor during the production process. This is smaller than the total bioreactor volume, and is typically about 10 L to about 50,000 L. Thus, the exemplary volumes described above for bioreactor volume are also applicable to the fill production process volume.

[0020] As described herein, a method for modulating afucosylation involves adding an amount of mannose to a bioreactor during the antibody production process. As used herein, "mannose" refers to a sugar monomer of the aldohexose family of carbohydrates and is the C-2 epimer of glucose. Mannose exists in both D- and L-isomer forms, but typically it is the D-isomer that is used in the methods of the present invention. [ka]

[0021] The methods described herein preferably include adding mannose to a bioreactor in which an antibody production process is taking place, thereby adding mannose itself to the antibody production process. The amount of mannose added (i.e., the weight of mannose) is calculated based on the fill volume of the bioreactor and thus the volume of the production process. Therefore, a mannose-containing solution is added to achieve a desired concentration in the cell culture medium. For example, 1 L of 10 g / L mannose feedstock can be added to a bioreactor containing 9 L of cell culture medium (medium and cell biomass) to achieve a final concentration of 1 g / L. Mannose addition can be carried out using any suitable process, including addition through one or more valves or ports on the bioreactor, adding mannose directly to the bioreactor through an opening or top of the bioreactor, or the mannose can be added to a solution added to the bioreactor. For example, mannose can be included in the nutrient medium solution introduced during the antibody production process, thereby also adding mannose to the process. It can be added as a standalone solution or as a multi-component medium feed. It should be noted that the methods described herein require the addition of mannose to the antibody production process in order to increase / control afucosylation levels, rather than simply including small amounts of mannose in the nutrient medium to promote cell growth, etc.

[0022] The amount of mannose added will depend on the degree of afucosylation required and the ability of the cells to tolerate the addition of mannose. Typically, the amount of mannose added is at least 1 g / L, for example, more than about 2 g / L, more than about 3 g / L, more than about 4 g / L, more than about 5 g / L, more than about 6 g / L, more than about 7 g / L, more than about 8 g / L, or more than about 9 g / L. The amount of mannose added is typically less than about 20 g / L, for example, less than about 15, 14, 13, 12, 11, or 10 g / L, or less than about 9 or 8 g / L, or less than about 7 g / L. Adding too much mannose can result in high osmotic pressure, which can in turn reduce cell growth. Therefore, the amount of mannose added can be about 1 g / L to about 20 g / L, about 1 g / L to about 15 g / L, 14 g / L, 13 g / L, 12 g / L, 11 g / L, 10 g / L, 9 g / L, 8 g / L, or 7 g / L, about 2 g / L to about 15 g / L, 14 g / L, 13 g / L, 12 g / L, 11 g / L, 10 g / L, 9 g / L, 8 g / L, or 7 g / L, or about 3 g / L to about 15 g / L, 14 g / L, 13 g / L, 12 g / L, 11 g / L, 10 g / L, 9 g / L, 8 g / L, or 7 g / L. Depending on cell production performance, greater or lesser amounts of mannose can also be added to achieve a desired afucosylation level. 1 g / L of mannose is equivalent to 5.6 mM mannose.

[0023] In one embodiment, if maximum fucosylation is desired, the amount of mannose added will typically be toward the higher levels outlined above. In another embodiment, if the goal is to match afucosylation to a target level of antibody with reference to a previous production process, the addition of mannose can be at a level in any of the ranges above, or can also be varied during the production process to achieve the desired matching level.

[0024] The timing of mannose addition to an antibody production process can vary based on the type of antibody being produced, the type of process being performed, the bioreactor, the requirements of the production plant in which the process is being performed, etc. Antibody production processes typically have a growth phase to rapidly achieve a desired cell density / biomass, followed by a production phase to promote high specific productivity of the antibody of interest. In some processes, the transition from the growth phase to the production phase is accompanied by a temperature shift.

[0025] To achieve modulation of afucosylation of the final product, mannose levels may be elevated during all or part of the production phase. This can be achieved by providing mannose-containing medium at various stages during the cell culture process. Typically, mannose is added during the entire production phase, but mannose can also be provided during the growth phase, and then the cells can be allowed to produce the antibody of interest under elevated mannose levels during the production phase without further mannose addition.

[0026] Production is typically one of two major production process methods known in the art: fed-batch and perfusion processes, or a hybrid of the two.

[0027] In some embodiments, mannose is added within (i.e., during) the first 12 to 48 hours of the cell culture process, more preferably within the first 12 to 36 hours, including within the first 12 hours, the first 24 hours, or the first 36 hours. In other embodiments, mannose is added toward the end of the growth phase, for example, 12 to 24 hours prior to the production phase. Mannose addition can also be made multiple times throughout the cell culture process (i.e., during the growth and / or production phases (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)), with each addition being in the desired amount (i.e., g / L amount) to achieve the final desired amount of mannose added.

[0028] In one embodiment, mannose is added in fixed amounts according to a predefined strategy, such as a recipe. For example, mannose may be included with one of the standard feeds and employ the same timing regimen, or it may be included as a separate feed.

[0029] In another embodiment, the amount of mannose added is adjusted as a result of periodic measurements of the expressed antibody, for example, if those measurements indicate that afucosylation deviates from a predetermined target value. The amount of mannose added is then increased if afucosylation is decreasing and decreased if afucosylation is increasing relative to the target value. Measurement of afucosylation can be performed offline or inline.

[0030] The predetermined value can be based on afucosylation levels obtained when the process was performed on a previous occasion, e.g., in a different size vessel, at a different site, etc. It can also be based on analysis of a commercially available product, so that the CQAs can be adhered to as closely as possible for regulatory purposes, e.g., when producing a biosimilar of that product. The predetermined level can be expressed, for example, as a range (e.g., 4.0-4.5%) or a midpoint with a tolerance (e.g., 4.25% + / - 0.25%).

[0031] As described herein, when the purpose of the process is to increase afucosylation as a result of the addition of mannose to the antibody production process, the afucosylation of the antibody product produced by the antibody production process is typically increased by at least 0.5% compared to the same antibody product when produced by the antibody production process without the addition of mannose. That is, when the antibody product from the production methods described herein (including the addition of mannose) is compared to the same antibody product produced in the same antibody production process but without the addition of additional mannose, the antibodies produced by the methods described herein exhibit at least 0.5% increased afucosylation.

[0032] Measurement of the amount of increased afucosylation of antibody products prepared according to the present methods involving the addition of mannose, compared to the same antibody products produced with the addition of mannose, can be performed using methods known in the art, such as mass spectrometry, including liquid chromatography-mass spectrometry, and other methods. Also, as noted above, there can be some variation between the methods used, so in one embodiment, the percentage is measured using a TOF LC / MS system, in which the antibody is reduced and then loaded directly into a liquid chromatograph mass spectrometer (LC-MS), such as an Agilent 6230B TOF LC / MS system (no PNGase F digestion required), and the reduced antibody is passed through a reverse-phase desalting column on the liquid chromatograph before being injected into the time-of-flight mass spectrometer.

[0033] Other suitable methods include those described, for example, in Tay and Butler, 2015, J. Biol. Methods 2:19; Mishra et al., 2020, J. Biotechnology X 5:100015. In one of these described methods, glycans can be removed using PNGase F and dried. They are then labeled using 2-AB labels and analyzed using hydrophilic interaction liquid chromatography-HPLC (HILIC-HPLC).

[0034] Comparison of the amount of afucosylation (or fucosylation) from one protein population to another provides a percentage increase in afucosylation (or decrease in fucosylation), generally provided for a population of antibodies. That is, measurements of the percent increase in afucosylation from one antibody population compared to another antibody population are generally calculated based on the amount of antibody produced, which is about 0.5 g / L or greater, rather than on individual antibodies. Preferably, the amount of antibody produced using the methods described herein is about 1 g / L or greater, preferably 5 g / L or greater, or about 10 g / L or greater. Thus, in embodiments herein where there is at least a 0.5% increase in afucosylation, the increase is measured relative to the total amount of antibody, which is about 0.5 g / L or greater.

[0035] In exemplary embodiments, the amount of increase in afucosylation resulting from the methods described herein is at least a 0.5% increase, or in other embodiments, at least a 0.6%, at least a 0.7%, at least a 0.8%, at least a 0.9%, at least a 1%, at least a 1.1%, at least a 1.2%, at least a 1.3%, at least a 1.4%, at least a 1.5%, at least a 1.6%, at least a 1.7%, at least a 1.8%, at least a 1.9%, at least a 2.0%, at least a 2.1%, at least a 2.2%, at least a 2.3%, at least a 2.4%, at least a 2.5%, at least a 2.6%, at least a 2.7%, at least a 2.8%, at least a 2.9%, at least a 3.0% increase, or at least a 0.5% to about 2.0% increase, about a 0.5% to about 1.5% increase, or about a 0.5% to about 1.0% increase.

[0036] In another embodiment, the process is used to control the fucosylation level to match a predetermined level (target value). Thus, in one embodiment, there is provided a method for matching the afucosylation of a recombinantly produced antibody to a target afucosylation percentage previously obtained for the same antibody, comprising culturing cells expressing the antibody in a bioreactor and controlling the addition of mannose to the bioreactor during the antibody production process to obtain an expressed antibody with a target afucosylation percentage.

[0037] Typically, afucosylation levels are controlled to within + / - 0.25% of the desired target value (if the target value is a range, the variation is relative to the midpoint of the range). For example, afucosylation levels are controlled to within + / - 0.5%.

[0038] The antibody production process used in the methods described herein is preferably carried out in mammalian cells, although in other embodiments, bacterial or insect cells may also be used to prepare antibody products. Exemplary mammalian cells that can be used in the antibody production process include human, mouse, rat, Chinese hamster, Syrian hamster, monkey, ape, dog, horse, ferret, and cat cells. In embodiments, the cells are Chinese hamster ovary (CHO) cells. In exemplary embodiments, the cells are CHO-K1 cells, CHOK1SV® cells, DG44 CHO cells, DUXB11 CHO cells, CHO-S, CHO GS knockout cells (CHO cells in which all endogenous copies of the glutathione synthetase (GS) gene have been inactivated), CHOK1SV® FUT8 knockout cells, CHOZN, or CHO-derived cells. An exemplary CHO GS knockout cell (e.g., GS-KO cell) is a CHOK1SV® GS knockout cell (e.g., GS Xceed® cells - CHOK1SV GS-KO®, Lonza Biologics, Inc., etc.). A CHO FUT8 knockout cell is, for example, Potelligent® CHOK1SV® FUT8 Knockout (Lonza Biologics, Inc.). In other embodiments, the cell may be derived from a mouse myeloma (NS0) cell line, HT1080, H9, HEK293 cell line, HeLa cell line, T cells, or cell lines such as HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, YO, C127, L cells, COS, etc.

[0039] In embodiments, the antibody production process is carried out in a fed-batch bioreactor, where a fed-batch production process is used, where nutrient medium is provided to the antibody production process and product remains in the bioreactor until the end of the production run. In such embodiments, the addition of mannose is also performed so that the antibody is not removed until the end of the production run.

[0040] In additional embodiments, the antibody production process is a perfusion process carried out in a perfusion bioreactor. Exemplary perfusion bioreactors for use in the methods described herein may include a fermenter, a stirred-tank reactor, or a wave-type bioreactor. In some cases, the perfusion bioreactor may include a hollow vessel or container comprising a bioreactor volume for receiving a cell culture in a fluid growth medium. In some cases, the perfusion bioreactor may be disposed in association with a rotatable shaft coupled to an agitator to agitate the cell culture. Perfusion bioreactors can be made from a variety of materials, such as stainless steel or other metals, polymers (e.g., rigid or flexible polymers), or any combination thereof. In one embodiment, the perfusion bioreactor may include various components and devices, such as baffles, spargers, gas supplies, heat exchangers, etc., that enable the cultivation and growth of cells within the cell culture. In embodiments in which a perfusion reaction (and perfusion bioreactor) is utilized, mannose may be added consistently throughout the process to maintain a desired level of added mannose.

[0041] In some embodiments, the perfusion process can be a steady-state perfusion production process. In such cases, the perfusion bioreactor can continuously receive an input medium, such as a nutrient medium, and optionally mannose, through at least an input port, while an output medium can be continuously removed from the perfusion bioreactor through at least an output port. The continuous introduction of one or more input components via the input medium and the continuous removal of output or other materials via the output medium can maintain a steady state or a pseudo-steady state within the cell culture contained within the bioreactor. For example, a steady-state condition can involve maintaining a relatively constant volume of cell culture and medium in which the cell culture is disposed. As an example, the volume of the cell culture can be maintained so that it does not fluctuate by more than, for example, 10%, 8%, 5%, or 3%. In some implementations, a steady-state perfusion production process can maintain a desired cell density within the perfusion bioreactor.

[0042] In embodiments, a perfusion bioreactor may include one or more ports, such as an input port and an output port. The input port may be configured to allow one or more input components to be supplied to the perfusion bioreactor. In some cases, the one or more input components (also referred to as one or more input materials) may include one or more nutrients, such as glucose, vitamins, lipids, mannose, etc. In some cases, the one or more input components may be delivered to the perfusion bioreactor via a liquid or other medium entering the perfusion bioreactor, in which case the medium may be referred to as an input medium. For example, the input medium may be a nutrient medium used by cells to grow or proliferate in the perfusion bioreactor. In one embodiment, the output port may be configured to allow materials to be removed from the perfusion bioreactor. For example, the removed materials may include waste, by-products, or other spent materials (also referred to as output materials). In some cases, such materials may be removed via a liquid or other medium exiting the perfusion bioreactor. Such medium may be referred to as an output medium. In some implementations, the desired biological product can be collected from the bioreactor through the same outlet port, or the bioreactor can have a separate port for collection of the biological product.

[0043] In a further embodiment, provided herein is a method for increasing afucosylation of a mAb antibody product, the method comprising: providing a mAb antibody production process preferably carried out in mammalian cells in a bioreactor, the production process having a volume of at least 50 L; adding mannose to the bioreactor during the mAb antibody production process in an amount greater than about 5 g / L; and producing a mAb antibody product via the antibody production process, wherein afucosylation of the mAb antibody product is increased by at least 2% compared to a mAb antibody product produced by the mAb antibody production process without the addition of mannose.

[0044] In an exemplary embodiment, the mAb antibody production process is carried out in a fed-batch bioreactor. In another embodiment, the antibody production process is carried out in a perfusion bioreactor. Preferably, the antibody production process is a perfusion process.

[0045] Once product biosynthesis by the producing cells has progressed to a satisfactory point, the product can be harvested, for example, by removing the medium and separating the supernatant from cells and cellular debris. The product can be subjected to one or more purification / processing steps to obtain a purified product, such as affinity chromatography, ion exchange chromatography, filtration, and / or viral inactivation. The product can also be combined with one or more pharmaceutically acceptable carriers, excipients, or diluents, for example, with one or more of a buffer, a surfactant, a stabilizer (such as trehalose, sucrose, glycerol), an amino acid (such as glycine, histidine, arginine), a metal ion / chelator, a salt, and / or a preservative, to produce a composition, such as a formulated pharmaceutical composition.

[0046] Example - Modulation of afucosylation with addition of mannose CHO GS-KO cells were cultured in a chemically defined culture medium supplemented with different concentrations of different chemicals to test the effect of those chemicals on the N-linked glycan profile of the product produced by the culture, which was a model IgG antibody. Experimental flow: 1. Prepare culture medium with supplemented chemicals 2. Cultivate cells in the medium from (1). 3. Harvesting the mAb produced in the culture and determining the glycan profile of the purified, reduced, and desalted mAb product 4. Determine the statistical impact of chemical supplements on glycan product quality

[0047] Preparation of medium containing supplemented chemicals: Chemicals supplemented to the medium include: -None (control) -Mannose -N-acetylmannosamine (ManNAc) + galactose

[0048] Concentrated stock solutions of mannose, ManNAc, and galactose were prepared and added to the culture medium. Specific volumes of the stock solutions were supplemented to the culture medium to produce the conditions in Table 1: [Table 1]

[0049] Cell culture: GS-KO cells were plated at 2 x 10 in aerated shake flasks containing medium from Table 1. 5 Cells were inoculated at a density of 1000 cells / mL. Cultures were grown for 5 days in a temperature, CO2, and humidity controlled incubator. Samples were taken daily to monitor the health of the cultures.

[0050] Product Collection and Glycan Measurement: The mAb product was harvested from day 5 shake flask cultures and purified via Protein A capture. The purified mAb was prepared for glycan analysis by reducing the mAb to separate the heavy and light chains. The reduced mAb was injected into an LC-MS, and the reduced mAb was desalted by passing it through a reverse-phase desalting column on the LC before injection into a time-of-flight mass spectrometer (TOF MS) (Agilent 6230B). Three injections per sample were performed for technical replicates.

[0051] Glycan Data Analysis and Statistical Analysis: The resulting LC-MS data were processed using ProteinMetrics software to report the relative abundance of each glycan species. Glycan species measured during processing included: G0, G0F, G1F, G2F, G1F + NeuAc, G2F + NeuAc, and G2F + 2NeuAc. The percent afucosylated mAb was calculated by dividing the total amount of afucosylated species by the sum of afucosylated and fucosylated species (G0 / G0F + G0). Using GraphPad Prism, Dunnett's post-hoc analysis was performed to determine whether mAbs produced in cultures fed mannose- or galactose / ManNAc-supplemented medium were significantly different from mAbs produced in cultures fed unsupplemented medium (control).

[0052] Results / Discussion Supplementation of the culture medium with mannose, but not with galactose and ManNAc, significantly increased the % afucosylated mAb. Ta. Increasing the concentration of mannose caused a concomitant increase in afucosylated species in a linear relationship. Supplementation with higher concentrations of ManNAc (10 μM) produced statistically different results compared to the control, with ManNAc slightly reducing afucosylation. [Table 2]

[0053] Illustrative Embodiments Embodiment 1 is a method for increasing afucosylation of a recombinantly expressed antibody in a bioreactor, the method comprising culturing cells expressing the antibody in a bioreactor and adding mannose to the cell culture during the antibody production process, such that afucosylation of the antibody is increased compared to the same antibody produced by the antibody production process without the addition of mannose. Embodiment 2 includes the method of embodiment 1, wherein the amount of mannose added is from about 1 g / L to about 10 g / L. Embodiment 3 includes the method of embodiment 1 or 2, wherein the bioreactor has a volume of at least 10 L. Embodiment 4 includes the method of any one of Embodiments 1 to 3, wherein the antibody is a monoclonal IgG. Embodiment 5 includes the method of any one of embodiments 1 to 4, wherein the cell is mammalian. Embodiment 6 includes the method of any of embodiments 1-5, wherein the afucosylation of the antibody is increased by at least 0.5% compared to an antibody produced by the antibody production process without the addition of mannose. Embodiment 7 includes the method of embodiment 6, wherein afucosylation of the antibody product is increased by at least 1%, e.g., at least 2%, compared to an antibody produced by the antibody production process without the addition of mannose. Embodiment 8 includes the method of any one of embodiments 1 to 7, wherein the antibody production process is a fed-batch process. Embodiment 9 includes the method of any one of embodiments 1 to 7, wherein the antibody production process is a perfusion process. Embodiment 10 includes the method of any of embodiments 1 to 9, further comprising isolating the expressed antibody and, optionally, subjecting the antibody to one or more purification steps. Embodiment 11 is a method for matching the afucosylation of a recombinantly produced antibody to a target afucosylation percentage previously obtained for the same antibody, the method comprising culturing cells expressing the antibody in a bioreactor and controlling the addition of mannose to the bioreactor during the antibody production process to obtain an expressed antibody with a target afucosylation percentage. Embodiment 12 includes the method of embodiment 11, wherein the afucosylation percentage of the expressed antibody is within ±0.25% of the target afucosylation percentage. Embodiment 13 includes the method of embodiment 11 or 12, wherein the antibody production process is a perfusion process.

[0054] These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art without departing from the spirit and scope of the present invention, which is particularly set forth in the appended claims. In addition, it should be understood that aspects of the various embodiments may be interchanged in whole or in part. Furthermore, those skilled in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention as further set forth in such appended claims.

Claims

1. 1. A method for increasing afucosylation of a recombinantly expressed antibody in a bioreactor, comprising: Culturing cells expressing the antibody in a bioreactor; adding mannose to a cell culture during an antibody production process, such that the afucosylation level of the antibody is at least 2% higher than the afucosylation level of the same antibody produced by the antibody production process without the addition of mannose.

2. 2. The method of claim 1, wherein the amount of mannose added is from about 1 g / L to about 10 g / L.

3. 3. The method of claim 1 or 2, wherein the bioreactor has a volume of at least 10 L.

4. The method of any one of claims 1 to 3, wherein the antibody is a monoclonal IgG.

5. The method of any one of claims 1 to 4, wherein the cell is mammalian.

6. 6. The method of any one of claims 1 to 5, wherein the afucosylation level of the antibody is at least 2.5% higher than the afucosylation level of an antibody produced by the antibody production process without the addition of mannose.

7. 7. The method of claim 6, wherein the afucosylation level of the antibody is at least 3% higher than the afucosylation level of an antibody produced by the antibody production process without the addition of mannose.

8. The method of any one of claims 1 to 7, wherein the antibody production process is a fed-batch process.

9. The method according to any one of claims 1 to 7, wherein the antibody production process is a perfusion process.

10. 10. The method of any one of claims 1 to 9, further comprising isolating the expressed antibody and optionally subjecting the antibody to one or more purification steps.

11. 1. A method for matching the afucosylation of a recombinantly produced antibody to a target afucosylation percentage previously obtained for the same antibody, comprising: Culturing cells expressing the antibody in a bioreactor; controlling the addition of mannose to the bioreactor during the antibody production process to obtain the expressed antibody with the target afucosylation percentage.

12. 12. The method of claim 11, wherein the afucosylation percentage of the expressed antibody is within ±0.25% of the target afucosylation percentage.

13. 13. The method of claim 11 or 12, wherein the antibody production process is a perfusion process.

Images