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    • 专利摘要:
    NET COMPOSITIONS UNDERSTANDING ANTIBODIES HIGHLY CONCENTRATED BY ULTRAFILTRATION AND ITS METHOD OF PREPARATION. The present invention relates to a method for preparing a composition comprising highly concentrated antibodies by ultrafiltration in batch concentration mode having a first constant feed rate and a second controlled feed rate. The invention also relates to compositions obtained by said method.
    公开号:BR112014004828B1
    申请号:R112014004828-2
    申请日:2012-08-31
    公开日:2021-03-09
    发明作者:Kelby Lau；Jean Bender；Saeko Tanaka；Rumiko Wakayama；Hidenari Yamada；Tomonori Isoda；Masayoshi Oh-Eda
    申请人:Chugai Seiyaku Kabushiki Kaisha；Genentech, Inc；
    IPC主号:
    专利说明:

    Technical Field
    [001] The present invention relates to the field of biological science, more specifically to the field of an antibody preparation. In particular, the present invention relates to a method for preparing a composition comprising highly concentrated antibodies by ultrafiltration. The method used in the present invention allows antibody therapies to reach formulations of high concentration at room temperature, such as above 100 g / L, preferably above 200 g / L, particularly preferably above 250 g / L. Background of the Technique
    [002] There is a growing demand for highly concentrated low-volume formulations of antibody therapies for subcutaneous administration, especially in the field of chronic disease therapy, to improve patient convenience and compliance by offering outpatient treatment.
    [003] For the production of antibody drug substance, ultrafiltration / diafiltration (UF / DF) is typically the final process step. Ultrafiltration is a membrane-based separation process that separates molecules in solution based on size. Diafiltration is a specific type of ultrafiltration in which an aqueous buffer is added to the retentate. In this step, the purified drug substance is concentrated and exchanged for the protein concentration and excipient composition necessary for the pharmaceutical product formulation.
    [004] The predominant technology used in the industry for the ultrafiltration / diafiltration (UF / DF) process is a form of tangential flow filtration (TFF) (see generally Shiloach J. et al., 1988, Van Reis R. et al., 2001). In this technology, the protein solution is recirculated under pressure, tangentially to an ultrafiltration membrane. This TFF approach works well for the drug substance at low to moderate concentrations and, in most cases, a UF / DF process for one antibody is highly adaptable to another antibody with minimal modifications. However, in situations with high protein concentrations, a series of technical challenges in the performance of the process came (see in general, Shire SJ. Et al., 2004, Luo R. et al., 2006, Shire SJ., 2009).
    [005] Obtaining high-concentration formulations by TFF technology can be difficult because highly concentrated protein solutions can lead to limited mass transfer due to reduced flow and possible membrane clogging (for example, see, Suki A. et al., 1984, 1986, Kim KJ. et al., 1992). Although this can be overcome by increasing the surface area of the membrane or replacing the membrane, it can lead to a lower yield. Another limitation is the high viscosity that can lead to high feed pressure, exceeding the upper limit of membrane integrity during the process (for example, see, Turker M. et al., 1987, Liu J. et al., 2005) . While implementing an appropriate formulation design such as increasing ionic strength or adding particular compounds can help reduce viscosity (for example, see, Liu J. et al., 2006), it can be a challenging exercise to reduce viscosity ensuring the composition of a stable formulation. In the situation where greater reductions in viscosity are required, it can be addressed by processing at elevated temperatures (for example, see, Winter C., 2009). However, in such cases, protein stability may be compromised by prolonged exposure to higher temperatures. . The problem to be solved by the present invention, therefore, is to provide a new processing method to achieve high protein concentration by manipulating other processing parameters. List of Citations Patent Literature
    [006] [PTL 1] Liu J, Shire SJ. Reduced-viscosity concentrated protein formulations. US Patent Dec. 15, 2006, US20070116700 A1
    [007] [PTL 2] Winter C. Process for concentration of antibodies and therapeutic products thereof. US patent Feb. 19, 2009, US20090214522 A1 Non-patent literature
    [008] [NPL 1] Kim KJ, Fane AG, Fell CJD, Joy DC. Fouling mechanisms of membranes during protein ultrafiltration, J. Membr. Sci. 68 (1992) 79.
    [009] [NPL 2] Liu J, Nguyen MDH, Andya JD, Shire SJ. Reversible selfassociation increases the viscosity of a concentrated monoclonal antibody in aqueous solution. J Pharm Sci (2005), 94: 1928-1940.
    [0010] [NPL 3] Luo R, Waghmare R, Krishnan M, Adams C, Poon E, Kahn D. High concentration UF / DF of a monoclonal antibody. Strategy for optimization and scale-up, BioProcess Int. 4 (2006) 44.
    [0011] [NPL 4] Shiloach J, Martin N, Moes H. Tangential flow filtration. Adv Biotechnol Process (1988), 8: 97-125.
    [0012] [NPL 5] Shire SJ, Shahrokh Z, Liu J. Challenges in the development of high protein concentration formulations. J Pharm Sci (2004), 93: 1390-1402.
    [0013] [NPL 6] Shire SJ. Formulation and manufacturability of biologics. Curr Opin Biotechnol (2009), 20: 708-714
    [0014] [NPL 7] Suki A, Fane AG, Fell CJD. Flux decline in protein ultrafiltration, J. Membr. Sci. 21 (1984) 269.
    [0015] [NPL 8] Suki A, Fane AG, Fell CJD. Modeling fouling mechanisms in protein ultrafiltration, J. Membr. Sci. 27 (1986) 181.
    [0016] [NPL 9] Turker M, Hubble J. Membrane fouling in a constant-flux ultrafiltration cell, J. Membr. Sci. 34 (1987) 267.
    [0017] [NPL 10] Van Reis R, Zydney A. Membrane separations in biotechnology.Curr Opin Biotechnol (2001), 12: 208-211. Summary of the Invention
    [0018] The industry standard technology for concentrating proteins on a manufacturing scale is ultrafiltration by the tangential flow. The key challenges for products with high final concentrations are to avoid clogging the membrane and overcome the high supply pressure.
    [0019] In general terms, the present disclosure describes the specific manipulation of process parameters of successful protein concentration, such as an antibody preparation, pharmaceutical formulations containing such a preparation and its use in human or animal therapy.
    [0020] In embodiments, the present disclosure provides a method in which a feed flow rate is maintained at a high flow rate until an optimal protein concentration is then reduced to a lower value to continue to concentrate more. For example, the concentration is carried out at a feed flow rate equal to or greater than 200 LMH until the retentate solution is concentrated at a protein concentration greater than 200 g / L, where a feed pressure reaches up to 85- 100% of the maximum specified supply pressure of an ultrafiltration membrane, so additional concentration is continued at a feed flow rate equal to or less than 120 LMH. The protein concentration attainable within the operational limits is higher than the conventional process comprising one step with a constant feed flow rate or two steps with a reduction in feed flow control with the first transition.
    [0021] The present disclosure also provides, in modalities, a more preferred method in which a feed flow rate is kept as high as possible until the end of the concentration process. For example, a feed flow rate is automatically controlled in order to maintain a feed pressure within 85-100% of the maximum specified feed pressure of an ultrafiltration membrane once the feed pressure reaches 85-100% of the specified maximum supply pressure of an ultrafiltration membrane under a constant feed flow rate.
    [0022] The present disclosure also provides, in modalities, the effectiveness of a stage of circulation inserted in the middle of a concentration process. For example, 20 minute circulation at a feed flow rate of 10-80 LMH is entered once the feed pressure reaches 85-100% of the specified maximum feed pressure of an ultrafiltration membrane under a feed rate of constant feeding. This circulation step can mitigate the increase in supply pressure during a subsequent ultrafiltration process.
    [0023] In summary, it is an object of the present invention to provide the following [1] to [33].
    [0024] [1] A method for preparing a composition comprising highly concentrated antibodies by ultrafiltration, wherein the method comprises the steps of:
    [0025] 1) regulation of a feed flow rate to allow the value of the feed pressure applied to an ultrafiltration membrane to increase to 85-100% of a specified maximum feed pressure of an ultrafiltration membrane; and
    [0026] 2) reduction of the feed flow rate to maintain or reduce the value of the feed pressure applied to the ultrafiltration membrane after step (1).
    [0027] [2] The method of [1], in which the antibody preparation is processed at room temperature.
    [0028] [3] The method of [1], in which the antibody preparation is processed at a temperature of 10 to 30 degrees C.
    [0029] [4] The method of [1], in which the antibody preparation is processed at a temperature of 15 to 30 degrees C.
    [0030] [5] The method of [1], in which highly concentrated antibodies have a high concentration above 100 g / L or a viscosity above 2 mPa.s.
    [0031] [6] The method of [1], in which highly concentrated antibodies have a high concentration above 200 g / L or a viscosity above 10 mPa.s.
    [0032] [7] The method of [1], in which highly concentrated antibodies have a high concentration above 250 g / L or a viscosity above 40 mPa.s.
    [0033] [8] The method of [1], in which the feed flow rate in step (1) is maintained at 200 LMH (L / m2 / hour) or higher.
    [0034] [9] The method of [1], in which the feed flow rate in step (1) is maintained at 250 LMH (L / m2 / hour) or higher.
    [0035] [10] The method of [1], [8] and [9], in which the feed flow rate in step (1) is maintained at a constant rate.
    [0036] [11] The method of [1], in which the maximum value of the supply pressure applied to an ultrafiltration membrane in step (1) is from 2.0 bar to 4.0 bar.
    [0037] [12] The method of [1], in which the maximum value of the supply pressure applied to an ultrafiltration membrane in step (1) is 3.5 bar.
    [0038] [13] The method of [1], wherein the maximum value of the supply pressure applied to an ultrafiltration membrane in step (1) is 85-100% of the specified maximum supply pressure of the ultrafiltration membrane.
    [0039] [14] The method of [1], in which step (1) is carried over to step (2) when the retentate solution is concentrated to a protein concentration greater than 200 g / L.
    [0040] [15] The method of [1], in which step (1) is carried over to step (2) when the retentate solution is concentrated to a protein concentration equal to or greater than 220 g / L.
    [0041] [16] The method of [1], in which step (1) is carried over to step (2) when the retentate solution is concentrated to a protein concentration equal to 240 g / L.
    [0042] [17] The method of [13], in which the feed flow rate after the feed pressure value is reduced in step (2) is maintained at a constant rate.
    [0043] [18] The method of [13] or [17], in which the feed flow rate after the supply pressure value is reduced in step (2) is maintained at 120 LMH (L / m2 / hour ) or lower.
    [0044] [19] The method of [13] or [17], in which the feed flow rate after the feed pressure value is reduced in step (2) is maintained at 80 LMH (L / m2 / hour ) or lower.
    [0045] [20] The method of [1], in which the value of the supply pressure applied to an ultrafiltration membrane in step (2) is kept at a constant value.
    [0046] [21] The method of [1], in which the value of the supply pressure applied to an ultrafiltration membrane in step (2) is kept within 85-100% of the specified maximum supply pressure of the ultrafiltration membrane by slowing down a feed flow rate.
    [0047] [22] The method of [20] or [21], in which the supply flow rate is automatically regulated in a way to maintain the supply pressure within 85-100% of the specified maximum supply pressure of the ultrafiltration membrane by a feedback control between a feed pressure and a feed flow rate.
    [0048] [23] The method of [1], further comprising between step (1) and step (2), the following step of:
    [0049] recirculation of the antibody preparation through the membrane with a closed permeate valve.
    [0050] [24] The method of [23], in which the antibody preparation is recirculated with a fully open retentate pressure control valve.
    [0051] [25] The method of [23], in which the feed flow rate in step (3) is maintained at a constant flow rate between 5 to 120 LMH (L / m2 / hour).
    [0052] [26] The method of [23], in which the feed flow rate in step (3) is maintained at a constant flow rate between 10 to 80 LMH (L / m2 / hour).
    [0053] [27] The method of [1], wherein the buffer composition of the antibody preparation is histidine between 10 and 30 mmol / L.
    [0054] [28] The method of [1], wherein the buffer composition of the antibody preparation is 20 mmol / L histidine.
    [0055] [29] The method of [1], wherein the pH of the antibody preparation is between pH 3.0 and pH 10.0.
    [0056] [30] The method of [1], wherein the pH of the antibody preparation is between pH 5.5 and pH 6.5.
    [0057] [31] The method of [1], wherein the pH of the antibody preparation is pH 6.0.
    [0058] [32] The method of [1], in which the ultrafiltration membrane has a molecular weight cut of 50 kDa or less.
    [0059] [33] The method of [1], in which the ultrafiltration membrane has a molecular weight cut of 30 kDa or less.
    [0060] [34] The method of [1], wherein the composition comprises highly concentrated human interleukin-6 anti-receptor monoclonal antibodies.
    [0061] [35] The method of [34], in which the composition comprises highly concentrated tocilizumab.
    [0062] [36] A liquid composition comprising highly concentrated antibodies prepared by the method of [1].
    [0063] [37] A liquid pharmaceutical composition comprising highly concentrated antibodies prepared by the method of [1] and a pharmaceutically acceptable carrier.
    [0064] [38] A method for preparing a composition comprising highly concentrated proteins by ultrafiltration, wherein the method comprises the steps of:
    [0065] 1) regulation of a feed flow rate to allow the value of the feed pressure applied to an ultrafiltration membrane to increase to 85-100% of a specified maximum feed pressure of an ultrafiltration membrane; and
    [0066] 2) reduction of the feed flow rate to maintain or reduce the value of the feed pressure applied to the ultrafiltration membrane after step (1).
    [0067] It will also be understood that both the preceding summary of the present invention and the following detailed description are of exemplified modalities, and not restrictive of the present invention or other alternative embodiments of the present invention. Other objects and attributes of the invention will become more fully evident when the following detailed description is read in conjunction with the accompanying figures and examples. In particular, although the invention is described in this application with reference to several specific embodiments, it will be appreciated that the description is illustrative of the invention and is not construed as limiting the invention. Various modifications and applications can occur to those skilled in the art, without departing from the spirit and scope of the invention, as described by the added claims. Likewise, other objects, attributes, benefits and advantages of the present invention will be evident from these summary and certain modalities described below and will be readily apparent to those skilled in the art. Such objects, attributes, benefits and advantages will be evident from the aforementioned together with the accompanying examples, data, figures and all reasonable inference to be drawn from them, alone or with consideration of the references incorporated in this application. Brief Description of Drawings
    [0068] Various aspects and applications of the present invention will become evident to the verse after considering the brief description of the figures and the detailed description of the present invention and its preferred modalities as follows:
    [0069] [fig. 1] Figure 1 shows an apparatus from the UF / DF process, in the modalities of the present disclosure.
    [0070] [fig. 2] Figure 2 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP on a laboratory scale. The feed flow rate was set at a constant rate of 250 LMH (L / m2 / hour) during the entire process.
    [0071] [fig. 3] Figure 3 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP on a laboratory scale. The feed flow rate was reduced to 80 LMH when the volume of retentate reached the value that corresponds to the protein concentration of 100 g / L.
    [0072] [fig. 4] Figure 4 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP on a laboratory scale. The feed flow rate was reduced to 80 LMH when the volume of retentate reached the value that corresponds to the protein concentration of 200 g / L.
    [0073] [fig. 5] Figure 5 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP on a laboratory scale. The feed flow rate was reduced to 80 LMH when the feed pressure exceeded 3.5 bar, which corresponded to a protein concentration of 240 g / L.
    [0074] [fig. 6] Figure 6 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP on a laboratory scale. Since the feed pressure exceeded 3.5 bar under a constant feed flow rate of 250 LMH, the feed flow rate was established in automatic flow control in a way to maintain the feed pressure of 3.5 Pub. The operation was terminated when the feed flow rate decreased to 80 LMH.
    [0075] [fig. 7] Figure 7 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP on a laboratory scale. The flow path has been changed in the circulation mode since the supply pressure has exceeded 3.5 bar. After circulating for 20 minutes at a constant feed flow rate of 80 LMH, ultrafiltration was resumed at the same feed flow rate.
    [0076] [fig. 8] Figure 8 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP on a laboratory scale. Circulation was carried out at a constant feed flow rate of 10 LMH.
    [0077] [fig. 9] Figure 9 summarizes the protein concentration of the group recovered on a laboratory scale, in terms of the present disclosure.
    [0078] [fig. 10] Figure 10 shows the viscosity profile of a humanized concentrated monoclonal antibody to IL-6R, in the modalities of the present disclosure.
    [0079] [fig. 11] Figure 11 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in stages of UF1 / DF / UF2 on the experimental scale.
    [0080] [fig. 12] Figure 12 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in stages UF3 / UF4 on an experimental scale.
    [0081] [fig. 13] Figure 13 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in stages UF1 / DF / UF2 on an experimental scale.
    [0082] [fig. 14] Figure 14 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in the UF3 / UF4 steps on an experimental scale.
    [0083] [fig. 15] Figure 15 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in steps UF1 / DF / UF2 on a manufacturing scale.
    [0084] [fig. 16] Figure 16 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in the UF3 / UF4 steps on a manufacturing scale.
    [0085] [fig. 17] Figure 17 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in the UF1 / DF / UF2 steps on a manufacturing scale.
    [0086] [fig. 18] Figure 18 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in the UF3 / UF4 steps on a manufacturing scale.
    [0087] [fig. 19] Figure 19 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP. The feed flow rate was operated at a constant rate of 250 LMH (L / m2 / hour) and then reduced to 80 LMH when the volume of retentate reached the value that corresponds to the protein concentration of 60 g / L.
    [0088] [fig. 20] Figure 20 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP. The feed flow rate was reduced to 80 LMH when the feed pressure exceeded 3.5 bar. The volume of retentate at that point corresponds to a protein concentration of 145 g / L.
    [0089] [fig. 21] Figure 21 shows the process values measured over time for the feed flow rate, feed pressure, retentate pressure, TMP. Since the feed pressure exceeded 3.5 bar under a constant feed flow rate of 250 LMH, the feed flow rate was established in automatic flow control in a way to maintain the feed pressure of 3.5 Pub. The operation was terminated when the feed flow rate decreased to 80 LMH. Description of Modalities
    [0090] Although any method and materials similar or equivalent to those described in this application can be used in the practice or testing of modalities of the present invention, the preferred methods are now described. However, before the present methods are described, it should be understood that the present invention is not limited to sizes, shapes, dimensions, materials, methodologies, particular protocols, etc. described in this application, as they may vary according to regular experimentation and optimization. It should also be understood that the terminology used in the description is for the purpose of describing the particular versions or modalities only, and is not intended to limit the scope of the present invention which will only be limited by the added claims.
    [0091] The disclosure of each publication, patent or patent application mentioned in this specification is specifically incorporated by reference in this application in its entirety. However, nothing in this application should be construed as an admission that the invention is not entitled to predate such disclosure by virtue of the previous invention.
    [0092] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by one of ordinary skill in the art to which the present invention belongs. In case of conflict, this specification, including definitions, will control. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.
    The present invention relates to a method for preparing a composition comprising highly concentrated antibodies by ultrafiltration.
    [0094] The present invention comprises a method for preparing a composition comprising highly concentrated antibodies by ultrafiltration in which a feed flow rate and a feed pressure applied to an ultrafiltration membrane are variable and modified during a filtration process.
    [0095] Particularly preferred embodiments of the present invention are shown below.
    [0096] A method for preparing a composition comprising highly concentrated antibodies by ultrafiltration, wherein the method comprises the steps of:
    [0097] regulation of a feed flow rate to allow the value of the feed pressure applied to an ultrafiltration membrane to increase to 85-100% of a specified maximum feed pressure of an ultrafiltration membrane; and
    [0098] reduction of the feed flow rate to maintain or reduce the value of the feed pressure applied to the ultrafiltration membrane after step (1).
    [0099] The term "ultrafiltration" which is used within the present invention denotes a membrane-based separation process that separates molecules in solution based on size. The term "tangential flow filtration (TFF)" denotes a specific filtration method in which a fluid flows tangentially to a membrane. The solution containing protein molecules is concentrated by flowing along, that is, tangential to the surface of an ultrafiltration membrane under pressure. The ultrafiltration membrane has a pore size with a certain cut-off value. In one embodiment, the cut-off value is in the range of 50 kDa or less, preferably 30kD or less.
    [00100] The term "feed flow" denotes the flow of fluid from the feed pump to the membrane. The term "feed flow rate" denotes the volume flow rate of the solution to the membrane. The feed flow rate is usually given in terms of volume per unit of time as liter / minute and normalized in terms of volume per membrane area of unit per unit of time as liter / m2 / h (LMH). The term "flow" denotes the normalized permeate flow through the membrane in terms of volume per membrane area of unit per unit time as liter / m2 / h (LMH).
    [00101] The term "supply pressure" denotes the pressure applied to the entrance of an ultrafiltration membrane. The expression "maximum supply pressure" denotes the maximum acceptable value of the supply pressure that is specified by a vendor as an ultrafiltration membrane product specification. The term "retentate pressure" denotes the pressure applied to the drain of an ultrafiltration membrane. The term "permeate pressure" denotes the pressure applied to the permeate side of the ultrafiltration membrane. The term "transmembrane pressure (TMP)" denotes the pressure that causes the fluid to be filtered through an ultrafiltration membrane. The value of TMP can be calculated as: TMP = (P feed + P retentate) / 2 - P permeate
    [00102] TMP is an average of the supply pressure and the retentate pressure in the case where the permeate side is open in the TFF equipment. The pressure value is usually given in terms of "bar" or "MPa" or "psi".
    [00103] The term "antibody" refers to a protein that specifically recognizes an antigen. The antibody can be monoclonal or polyclonal. The antibody can exist in a variety of formats, including, for example, Fv, Fab and F (ab) 2 as well as single chain (scFv) or diabodies. In addition, any fragment or modification (e.g., chimeric antibody, humanized antibody, etc.) of antibody can be used for the present method. Methods for preparing these types of antibodies are well known in the art, and any method can be employed in the present invention to prepare such antibodies and fragments thereof.
    [00104] The monoclonal antibodies used in the present invention include not only those derived from animals such as humans, mice, rats, hamsters, rabbits, sheep, camels, and monkeys, but also artificially genetically modified recombinant antibodies such as chimeric antibodies, humanized antibodies and bispecific antibodies. The antibodies of the present invention also include genetically recombinant antibodies that result from artificially modifying constant regions of the antibody to alter the physical properties of the antibody molecule (specifically, changing the isoelectric point (pI), improving affinity for the Fc receptor, etc.) for the purpose of improving blood persistence and in vivo pharmacokinetics.
    [00105] The immunoglobulin class of antibodies used in the present invention is not particularly limited; and the class can be any class, including IgG such as IgG1, IgG2, IgG3, and IgG4, IgA, IgD, IgE and IgM. However, IgG and IgM are preferred.
    [00106] The antibodies used in the present invention include, but are not limited to tissue anti-factor antibodies, anti-IL-6 receptor antibodies, anti-IL-6 antibodies, HM1.24 anti-antigen antigen antigens, parathyroid hormone-related antibodies (antibodies anti-PTHrP), anti-glypican-3 antibodies, anti-GM3 antibodies, TPO receptor antiagonist antibodies, antibodies as a functional substitute for coagulation factor VIII, anti-IL31 receptor antibodies, anti-HLA antibodies, anti-AXL antibodies, antibodies anti-CXCR4, anti-NR10 antibodies and bispecific antibodies against factor IX and factor X.
    Preferred humanized antibodies used in the present invention include humanized interleukin-6 (IL-6) anti-receptor antibodies (tocilizumab, hPM-1 and MRA) (see WO 92/19759 humanized HM1.24 anti-antigen antigens (see WO 98 / 14580), humanized anti-peptide antibodies related to parathyroid hormone (anti-PTHrP antibodies) (see WO 98/13388), humanized tissue antifactor antibodies (see WO 99/51743), humanized anti-glypican-3 antibodies IgG1kappa (see PCT / JP05 / 013103), and humanized anti-NR10 antibodies (see WO2009 / 072604) The particularly preferred humanized antibodies used in the present invention are the humanized IL-6 receptor antibodies.
    Preferred human IgM antibodies include recombinant human IgM anti-ganglioside antibodies GM3 (see WO 05/05636).
    Preferred minibodies include TPO receptor antiagonist bodies (see WO 02/33072) and CD47 antiagonist bodies (see WO 01/66737).
    In addition, antibodies with an improved isoelectric point include, for example, Mab1 which is an IL-6 antireceptor antibody described in WO 2011/090088 (chain H / SEQ ID NO: 1 in this; chain L / SEQ ID NO: 2 in this), and fully humanized NS22 antibody, which is a humanized anti-NR10 antibody, produced by the method described in Example 12 of WO2009 / 072604.
    [00111] The present invention also relates to a method for preparing a composition comprising highly concentrated proteins in addition to antibodies by ultrafiltration. The present invention comprises a method for preparing a composition comprising highly concentrated proteins by ultrafiltration in which a feed flow rate and a feed pressure applied to an ultrafiltration membrane are variable and modified during a filtration process. The proteins used in the present invention include, but are not limited to, enzymes, cytokines and peptide aptamers.
    [00112] The term "a composition comprising highly concentrated antibodies" as used within the present patent application denotes an aqueous, buffered solution containing the highly concentrated antibodies. The term "buffer", as used within the present patent application, denotes a solution in which changes in pH due to the addition or release of acidic or basic substances are leveled by a buffer substance. Any buffer substance that results in such an effect can be used. In one embodiment, pharmaceutically acceptable buffer substances are used, such as, for example, phosphoric acid or salts thereof, acetic acid or salts thereof, citric acid or salts thereof, morpholine or salts thereof, 2- (N-morpholino) ethanesulfonic acid or salts thereof, or tris (hydroxymethyl) aminomethane (TRIS) or salts thereof. In a preferred embodiment, the buffer composition of the antibody preparation is histidine between 10 and 30 mmol / L. In the most preferred embodiment the buffer composition of the antibody preparation is 20 mmol / L histidine.
    [00113] Optionally, the buffered solution can comprise an additional salt, such as, for example, sodium chloride, and / or sodium sulfate, and / or potassium chloride, and / or potassium sulfate, and / or sodium citrate and / or potassium citrate.
    [00114] In an embodiment of the present invention, the pH of the antibody preparation is between pH 3.0 and pH 10.0, preferably between pH 5.5 and pH 6.5, more preferably pH 6.0.
    [00115] In an embodiment of the present invention, the antibody preparation is processed at room temperature, preferably at a temperature of 10 to 30 degrees C, more preferably at a temperature of 15 to 30 degrees C.
    [00116] In one embodiment, highly concentrated antibodies have a protein concentration above 100 g / L or a viscosity above 2 mPa.s. In a preferred embodiment, highly concentrated antibodies have a protein concentration above 200 g / L or a viscosity above 10 mPa.s. In the most preferred embodiment, highly concentrated antibodies have a protein concentration above 250 g / L or a viscosity above 40 mPa.s.
    [00117] In one embodiment, the feed flow rate in step (1) is maintained at 200 LMH (L / m2 / hour) or higher. In a preferred embodiment, the feed flow rate in step (1) is maintained at 250 LMH (L / m2 / hour) or higher. In these embodiments, the feed flow rate in step (1) is preferably maintained at a constant rate.
    [00118] In one embodiment, the maximum supply pressure applied to an ultrafiltration membrane in step (1) is within 85-100% of the specified maximum supply pressure of the ultrafiltration membrane. In a preferred embodiment, the maximum supply pressure is 2.0 bar to 4.0 bar. In a more preferred embodiment, the maximum supply pressure applied to an ultrafiltration membrane in step (1) is 3.5 bar.
    [00119] In one embodiment, step (1) is carried over to step (2) when the retentate solution is concentrated to a protein concentration greater than 200 g / L. In a preferred embodiment, step (1) is carried over to step (2) when the retentate solution is concentrated to a protein concentration equal to or greater than 220 g / L. In a more preferred embodiment, step (1) is carried over to step (2) when the retentate solution is concentrated to a protein concentration equal to 240 g / L.
    [00120] In this mode, the feed flow rate after the feed pressure value is reduced in step (2) is maintained at a constant rate, preferably 120 LMH (L / m2 / hour) or less or more preferably 80 LMH (L / m2 / hour) or less.
    [00121] In one embodiment, the value of the supply pressure applied to the ultrafiltration membrane in step (2) is maintained at a constant value.
    [00122] In one embodiment, the value of the supply pressure applied to the ultrafiltration membrane in step (2) is maintained within 85-100% of the maximum specified supply pressure of the ultrafiltration membrane by slowing the flow rate of the feed.
    [00123] In one embodiment, the feed flow rate is automatically regulated in a way to maintain the feed pressure within 85-100% of the maximum specified feed pressure of the ultrafiltration membrane by a feedback control between a feed pressure. feed and a feed flow rate.
    [00124] In a modality of the production method, according to the present invention, it also comprises between stage (1) and stage (2), the following stage of:
    [00125] recirculation of the antibody preparation across the membrane with a closed permeate valve.
    [00126] In this embodiment, the antibody preparation is recirculated with a fully open retentate pressure control valve.
    [00127] In this mode, the feed flow rate in step (3) is preferably maintained at a constant flow rate between 5 and 120 LMH (L / m2 / hour), and more preferably between 10 and 80 LMH (L / H) m2 / hour).
    [00128] The present invention also relates to a liquid composition comprising highly concentrated antibodies prepared by the methods of the present invention.
    [00129] The present invention also relates to liquid pharmaceutical compositions. The liquid pharmaceutical compositions of the present invention can include pharmaceutically acceptable carriers.
    [00130] In the present invention, liquid pharmaceutical compositions ordinarily refer to agents for treating, preventing, testing or diagnosing diseases.
    [00131] The liquid pharmaceutical compositions of the present invention can be formulated by methods known to those skilled in the art. For example, they can be used parenterally, in the form of injections of sterile solutions or suspensions including water or other pharmaceutically acceptable liquid. For example, such liquid compositions can be formulated by mixing in the form of a unit dose required in approved manufacturing practice in general for medicines by appropriately combining with pharmaceutically acceptable vehicles or media, specifically with sterile water, physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder or similar. In such formulations, the amount of the active ingredient is adjusted to obtain an appropriate amount in a predetermined range.
    [00132] Sterile compositions for injection can be formulated using transports such as distilled water for injection, according to standard formulation practice.
    [00133] Aqueous injection solutions include, for example, physiological saline and isotonic solutions containing dextrose or other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol and sodium chloride). It is also possible to use in combination suitable solubilizers, for example, alcohols (ethanol and such), polyalcohols (propylene glycol, polyethylene glycol and the like), non-ionic surfactants (polysorbate 80 (TM), HCO-50 and the like).
    [00134] Oils include sesame oil and soy oils. Benzyl benzoate and / or benzyl alcohol can be used in combination as solubilizers. It is also possible to combine buffers (for example, phosphate buffer and sodium acetate buffer), sedative agents (for example, procaine hydrochloride), stabilizers (for example, benzyl alcohol and phenol), and / or antioxidants. Appropriate ampoules are filled with the prepared injections.
    [00135] The liquid pharmaceutical compositions of the present invention are preferably administered parenterally. For example, liquid compositions can be in dosage form for injections, transnasal administration, transpulmonary administration or transdermal administration. For example, they can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection or the like.
    [00136] The methods of administration can be appropriately selected in consideration of the age and symptoms of the patient. The dose of a liquid pharmaceutical composition containing an antigen binding molecule can be, for example, from 0.0001 to 1000 mg / kg for each administration. Alternatively, the dose can be, for example, from 0.001 to 100,000 mg per patient. However, the present invention is not limited by the numerical values described above. Doses and methods of administration vary depending on weight, age, symptoms of the patient and the like. Those skilled in the art can establish appropriate doses and methods of administration in consideration of the factors described above. EXAMPLES
    [00137] The following examples serve to more fully describe the way to use the disclosure described above, as well as to present the best ways contemplated to carry out various aspects of the disclosure. It is understood that these examples in no way serve to limit the true scope of this disclosure, instead they are presented for illustrative purposes. Comparative example 1
    [00138] FIG. 1 illustrates the main components of an apparatus used to carry out an ultrafiltration process. A recycling tank contains the starting material and the retentate. A mixing device ensures uniform mixing between the initial grouping added via a transfer line and the retentate that returns back to the ultrafiltration membrane recycling tank. A feed pump creates the tangential flow over the membrane. The supply pressure is measured at the inlet of the membrane. A retentate pressure control valve is used on the retentate side, downstream of the membrane, to adjust a retentate pressure, for example, under transmembrane pressure control (TMP). Between the membrane and the retentate pressure control valve, a pressure sensor measures a pressure of the retentate. On the permeate side of the membranes, a pressure of the liquid filtered through the membrane is monitored by a permeate pressure sensor.
    [00139] For laboratory scale ultrafiltration processing, an AKTAcrossflow automated TFF system (GE Healthcare, US) was used. The ultrafiltration process was performed using a 0.02 m2 Sartocon slide cassette with a regenerated cellulose Hydrosart membrane, a cut of nominal molecular weight of 30 kDa and a maximum feed pressure specification of 4.0 bar (Sartorius, Germany ).
    [00140] Before use, the membrane cassette was cleaned with 1 mol / L sodium hydroxide and rinsed with purified water. The normalized flow was determined to ensure comparable membrane properties. The membrane cassette was equilibrated with 30 mmol / L histidine buffer pH 5.8 prior to the process. Ultrafiltration was performed at room temperature.
    [00141] The starting material was prepared from a purified pool of a humanized human interleukin-6 anti-receptor monoclonal antibody (IL-6R) (tocilizumab (registered trademark: ACTEMRA, RoACTEMRA) see PCT Pub. No. WO92 / 19759, US Pat. No. 5795965). The purified pool was concentrated to 60 mg / mL and buffer exchanged with 30 mmol / L pH 5.8 histidine buffer.
    [00142] The buffer exchange pool (DF pool) was loaded into the TFF system with 625 g of antibody / m2. The feed flow rate was set at a constant rate of 250 LMH (L / m2 / hour) during the entire process. The TMP was controlled at 1.0 bar until the retentate pressure control valve was fully open. The ultrafiltration process was operated with the open point permeate side. The operation was terminated when the supply pressure exceeded 3.5 bar. After ultrafiltration processing, the concentrated solution was circulated with the permeate side closed for 15 minutes under a constant retentate flow rate of 10 mL / min and then recovered in a graduated cylinder. The recovered group was agitated until it was visually homogeneous.
    [00143] For the measurement of protein concentration, the recovered cluster was diluted gravimetrically using a density value measured by a DMA 4500 densitometer (Anton Paar, Austria). UV absorbance at 280 nm was measured with a UV / Vis DU800 spectrophotometer (Beckman Coulter, US).
    [00144] FIG. 2 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP. Table 1 shows the result of the protein concentration measurement.
    Comparative example 2
    [00145] Comparative Example 1 was repeated with the following exception. The feed flow rate was reduced to 80 LMH when the volume of retentate reached the value that corresponds to the protein concentration of 100 g / L.
    [00146] FIG. 3 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP. Table 2 shows the result of the measurement of protein concentration.
    Comparative example 3
    [00147] Comparative Example 1 was repeated with the following exception. The feed flow rate was reduced to 80 LMH when the volume of retentate reached the value that corresponds to the protein concentration of 200 g / L.
    [00148] FIG. 4 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP. Table 3 shows the result of the measurement of protein concentration.
    Example 4
    [00149] Comparative Example 1 was repeated with the following exception. The feed flow rate was reduced to 80 LMH when the feed pressure exceeded 3.5 bar. The volume of retentate at that point corresponds to a protein concentration of 240 g / L.
    [00150] FIG. 5 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP. Table 4 shows the result of the measurement of protein concentration.
    Example 5
    [00151] Example 4 was repeated with the following exception. Since the feed pressure exceeded 3.5 bar under a constant feed flow rate of 250 LMH, the feed flow rate was established in automatic flow control in a way to maintain the feed pressure of 3.5 Pub. The operation was terminated when the feed flow rate decreased to 80 LMH.
    [00152] FIG. 6 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP. Table 5 shows the result of the measurement of protein concentration.
    Example 6
    [00153] Example 4 was repeated with the following exception. The flow path was changed in the circulation mode since the supply pressure exceeded 3.5 bar. In the circulation mode, the retentate was circulated through the membrane with the retentate pressure control valve fully open and the permeate closed. After circulating for 20 minutes at a constant feed flow rate of 80 LMH, ultrafiltration was resumed at the same feed flow rate.
    [00154] FIG. 7 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP. Table 6 shows the result of the measurement of protein concentration.
    Example 7
    [00155] Example 6 was repeated with the following exception. Circulation was carried out at a constant feed flow rate of 10 LMH.
    [00156] FIG. 8 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP. TABLE 7 shows the result of the measurement of protein concentration.

    [00157] FIG. 9 summarizes the concentration of the pool recovered in Examples 1 to 7. Example 8
    [00158] The viscosity of a concentrated cluster of a humanized monoclonal antibody for IL-6R was measured using an AR1000 rheometer and 40 mm cone and plate geometry, 2 degree angle and 53 micrometer truncation (TA Instruments, USA).
    [00159] FIG. 10 shows the viscosity graph against concentration at temperatures of 15 degrees C, 25 degrees C and 35 degrees C. Comparative example 9
    [00160] For a scale study, the UF / DF process was carried out on an experimental scale. The process was operated in two stages with different sizes of the TFF system. A larger TFF system, using 1.20 m2 Sartocon cassettes, was used to process the UF1 / DF / UF2 steps. A smaller TFF system, using 0.30 m2 Sartocon cassettes, was used to process the UF3 / UF4 steps. The entire process was operated at room temperatures with the permeate side open. The Sartocon cassettes used were 30 kDa (cut) membranes from Hydrosart (Sartorius, Germany).
    [00161] Before use, the membrane cassettes were cleaned with 1 mol / L sodium hydroxide and rinsed with purified water. The normalized flow was determined to ensure comparable membrane properties.
    [00162] Before the process, the membrane cassettes were equilibrated with histidine buffer 30 mmol / L pH 5.8 in the large system and histidine buffer 20 mmol / L pH 6.1 in the small system respectively. The entire process was carried out at room temperature.
    [00163] In the large system, a purified pool of a humanized human anti-IL-6R monoclonal antibody was loaded with 259 g of antibody / m2. The feed flow rate was set at a constant rate of 710 LMH. The TMP was controlled at 1.0 bar. The purified pool was concentrated at 20 g / L in the UF1 step and then diafiltered with 7 diavolumes of 30 mmol / L histidine buffer pH 5.8. After diafiltration, the cluster was still concentrated at 60 g / L in the UF2 stage. The UF2 pool was circulated through the membrane for 15 minutes under a low differential pressure of 34.5 kPa (5 psi) and then recovered in a separate container.
    [00164] In the small system, the UF2 pool recovered was loaded with 990 g of antibody / m2. In the UF3 stage, the feed flow rate was established at a constant rate of 250 LMH. The UF3 stage was completed when the volume of retentate reached the value that corresponds to the protein concentration of 100 g / L. The feed flow rate was established at a constant rate of 80 LMH in the UF4 stage. The TMP was controlled at 1.0 bar until the retentate pressure control valve was fully open. The operation was completed when the volume of retentate decreased to the value that corresponds to the protein concentration of 240 g / L. It is noteworthy that the feed flow rate was manually reduced after 80 minutes as the feed pressure approached the upper limit before the retentate volume reached the target volume.
    [00165] The UF4 pool was circulated through the membrane for 15 minutes under a low differential pressure of 103.4 kPa (15 psi) and then recovered in a separate container. The pool of UF4 recovered was well mixed by inversion of the container.
    [00166] For the measurement of protein concentration, the recovered UF4 cluster was diluted gravimetrically using a density value measured by a Densito 30PX densitometer (Mettler Toledo, Switzerland). The UV absorbance at 280 nm was measured with a UV / Vis UV-1700 spectrophotometer (Shimadzu, Japan).
    [00167] FIG. 11 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in the steps of UF1 / DF / UF2.
    [00168] FIG. 12 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP in the stages of UF3 / UF4.
    [00169] TABLE 8 shows the result of the measurement of protein concentration.

    [00170] TABLE 9 shows the result of the step yield calculation.
    Example 10
    [00171] Comparative Example 9 was repeated with the following exceptions. The UF3 / 4 stages were performed using 0.40 m2 Sartocon cassettes with a 30 kDa cut Hydrosart membrane (Sartorius, Germany). In the large system, the purified pool was loaded with 274 g of antibody / m2. In the small system, the UF2 pool recovered was loaded with 804 g of antibody / m2. The process moved from the UF3 stage to the UF4 stage when the volume of retentate reached the value that corresponds to the protein concentration of 220 g / L.
    [00172] FIG. 13 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in the steps of UF1 / DF / UF2.
    [00173] FIG. 14 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in the stages of UF3 / UF4.
    [00174] Table 10 shows the result of the measurement of protein concentration.

    [00175] Table 11 shows the result of the step yield calculation.
    Example 11
    [00176] Example 10 was repeated with the following exceptions. TFF production scale systems were used in a GMP production facility. The UF1 / DF / UF2 steps were performed using 35.10 m2 Sartocon cassettes and the UF3 / 4 steps used 17.55 m2 Sartocon cassettes with a 30 kDa cut Hydrosart membrane (Sartorius, Germany). In a large system, the purified pool was loaded with 243 g of antibody / m2. In the small system, the pool of UF2 recovered was loaded with 478 g of antibody / m2. The DF buffer was changed to 39 mmol / L histidine buffer pH 5.8. The target protein concentration of the UF2 cluster was increased to 75 g / L. At the end of the UF2 stage, the feed flow rate was reduced to prevent foam in the recycle tank. To maximize recovery, the UF2 cluster and the UF4 cluster were recovered by removing 70 L and 1 L buffers respectively. The recovered UF4 pool was formulated at 180 g / L in 20 mmol / L histidine buffer pH 6.0, 30 mmol / L methionine, 100 mmol / L arginine and 0.2% polysorbate 80 (see PCT Pub. No. WO 2009/084659). For the measurement of protein concentration, the UF4 cluster and the recovered UF4 cluster were diluted gravimetrically using a density reference. The UV absorbance at 280 nm was measured with a UV / Vis UV-2450 spectrophotometer (Shimadzu, Japan).
    [00177] FIG. 15 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in the steps of UF1 / DF / UF2.
    [00178] FIG. 16 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in the stages of UF3 / UF4.
    [00179] Table 12 shows the result of the measurement of protein concentration.

    [00180] Table 13 shows the result of the step yield calculation.

    [00181] Histidine concentration was measured using an Alliance 2695 HPLC system (Waters, USA) and a YMC-Pack ODSA column, 250 x 4.6 mm (YMC, Japan). Table 14 shows the result of the histidine quantification assay.

    [00182] The monomer contents in the groupings in the process were measured using an Alliance 2695 HPLC system (Waters, USA) and a TSK G3000SWXL column (Tosoh, Japan). Table 15 shows the result of the SEC test.
    Example 12
    [00183] Example 11 was repeated with the following exception. In the large system, the purified pool was loaded with 246 g of antibody / m2. In the small system, the UF2 pool recovered was loaded with 482 g of antibody / m2.
    [00184] FIG. 17 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP and retentate volume in steps of UF1 / DF / UF2.
    [00185] FIG. 18 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP in the stages of UF3 / UF4.
    [00186] Table 16 shows the result of the measurement of protein concentration.


    [00187] Table 17 shows the result of the step yield calculation.

    [00188] Histidine concentration was measured using an Alliance 2695 HPLC system (Waters, USA) and a YMC-Pack ODSA column, 250 x 4.6 mm (YMC, Japan). Table 18 shows the result of the histidine quantification assay.

    [00189] The monomer contents in the process groupings were measured using an Alliance 2695 HPLC system (Waters, USA) and a TSK G3000SWXL column (Tosoh, Japan). Table 19 shows the result of the SEC test.
    Comparative example 13
    [00190] An automated AKTAcrossflow TFF system on a laboratory scale (GE Healthcare, US) was used for ultrafiltration processing. The ultrafiltration process was carried out using two 88 cm2 Pellicon 3 cassettes with Ultracel regenerated cellulose membranes, a cut of nominal molecular weight of 30 kDa (Merck Millipore, Germany).
    [00191] Before use, the membrane cassettes were cleaned with 0.5 mol / L sodium hydroxide and rinsed with purified water. The normalized flow was determined to ensure comparable membrane properties. The membrane cassettes were equilibrated with 20 mmol / L tris, 150 mmol / L arginine buffer pH 7.0 before the process. Ultrafiltration was performed at room temperature.
    [00192] The starting material was prepared from a purified pool of a humanized anti-NR10 monoclonal antibody (fully humanized NS22 antibody prepared according to the method shown in Example 12 of WO 2009/072604) which belongs to the IgG2 antibody class. This is an antibody whose amino acid sequence has been modified such that the pI is reduced to 5.6. The purified pool was concentrated to 20 mg / ml and buffer changed to tris 20 mmol / L, arginine buffer 150 mmol / L pH 7.0.
    [00193] The buffer exchange pool (DF pool) was loaded with 625 g of antibody / m2. The feed flow rate was operated at a constant rate of 250 LMH (L / m2 / hour) and then reduced to 80 LMH when the volume of retentate reached the value that corresponds to the protein concentration of 60 g / L. The TMP was controlled at 1.0 bar until the retentate pressure control valve was fully open. The ultrafiltration process was operated with the open point permeate side. The operation was terminated when the supply pressure exceeded 3.5 bar. After ultrafiltration processing, the concentrated solution was circulated with the permeate side closed for 15 minutes at a constant feed flow rate of 10 mL / min and then recovered in a graduated cylinder. The recovered group was agitated until it was visually homogeneous.
    [00194] For the measurement of protein concentration, the recovered cluster was diluted gravimetrically using a density value measured by a densitometer DMA 4500 (Anton Paar, Austria). UV absorbance at 280 nm was measured with a UV / Vis DU800 spectrophotometer (Beckman Coulter, US).
    [00195] FIG. 19 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP. TABLE 20 shows the result of the measurement of protein concentration.
    Example 14
    [00196] Comparative Example 13 was repeated with the following exception. The feed flow rate was reduced to 80 LMH when the feed pressure exceeded 3.5 bar. The volume of retentate at that point corresponds to a protein concentration of 145 g / L.
    [00197] FIG. 20 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP. TABLE 21 shows the result of the measurement of protein concentration.
    Example 15
    [00198] Example 14 was repeated with the following exception. Since the feed pressure exceeded 3.5 bar under a constant feed flow rate of 250 LMH, the feed flow rate was established in automatic flow control in a way to maintain the feed pressure of 3.5 Pub. The operation was terminated when the feed flow rate decreased to 80 LMH. [00199] FIG. 21 shows the process values measured over time by the feed flow rate, feed pressure, retentate pressure, TMP. TABLE 22 shows the result of the protein concentration measurement.
    权利要求:
    Claims (15)
    [0001]
    1. Method for preparing a composition comprising highly concentrated proteins or antibodies by ultrafiltration, characterized by the fact that it comprises the steps of: concentrating an antibody or protein preparation by ultrafiltration of the preparation, said ultrafiltration comprising: (1) regulation of the rate feed flow of the preparation to allow the value of the feed pressure applied to an ultrafiltration membrane to increase to 85-100% of a specified maximum feed pressure of an ultrafiltration membrane; and (2) reduction of the feed flow rate to maintain or reduce the value of the feed pressure applied to the ultrafiltration membrane after step (1), where the feed flow rate in step (1) is maintained at 250 LMH (L / m2 / hour) or higher, and the feed flow rate in step (2) is reduced to 80 LMH (L / m2 / hour) or lower, where the preparation of highly concentrated antibodies or proteins directly after said ultrafiltration has a concentration of antibodies or proteins above 200 g / L or a viscosity above 10 mPa ^ s, where ultrafiltration is a form of tangential flow filtration (TFF), and in which the maximum pressure value of feeding applied to an ultrafiltration membrane in step (1) is 350 kPa (3.5 bar) to 400 kPa (4.0 bar).
    [0002]
    2. Method according to claim 1, characterized by the fact that the antibody or protein preparation is ultrafiltered at a temperature of 15 to 30 ° C.
    [0003]
    Method according to claim 1 or 2, characterized by the fact that the concentrated preparation has a concentration above 250 g / L or a viscosity above 40 mPa ^ s.
    [0004]
    Method according to any one of claims 1 to 3, characterized in that the feed flow rate in step (1) is maintained at a constant rate.
    [0005]
    Method according to any one of claims 1 to 4, characterized by the fact that step (1) is carried over to step (2) when the retentate solution is concentrated to a protein concentration greater than 200 g / L , equal to or greater than 220 g / L, or equal to 240 g / L.
    [0006]
    Method according to any one of claims 1 to 5, characterized in that the flow rate of the feed after the feed pressure value is reduced in step (2) is maintained at a constant rate.
    [0007]
    Method according to any one of claims 1 to 6, characterized in that the value of the supply pressure applied to an ultrafiltration membrane in step (2) is maintained at a constant value, or within 85-100 % of the maximum specified feed pressure of the ultrafiltration membrane reducing the feed flow rate.
    [0008]
    8. Method according to claim 7, characterized in that the feed flow rate is automatically regulated in a way to maintain the feed pressure within 85-100% of the specified maximum feed pressure of the ultrafiltration membrane by a feedback control between a supply pressure and a feed flow rate.
    [0009]
    Method according to any one of claims 1 to 8, characterized in that it further comprises between step (1) and step (2), the following step of: (3) recirculation of the antibody or protein preparation through the membrane with a closed permeate valve.
    [0010]
    10. Method according to claim 9, characterized in that the antibody or protein preparation is recirculated with a fully open retentate pressure control valve.
    [0011]
    11. Method according to claim 9, characterized by the fact that the feed flow rate in step (3) is maintained at a constant flow rate between 5 and 120 LMH (L / m2 / hour), or between 10 and 80 LMH (L / m2 / hour).
    [0012]
    12. Method according to any one of claims 1 to 11, characterized in that the buffer composition of the antibody or protein preparation is histidine between 10 and 30 mmol / L, or 20 mmol / L of histidine.
    [0013]
    13. Method according to any one of claims 1 to 12, characterized in that the pH of the antibody preparation is between pH 5.5 and pH 6.5, or is pH 6.0.
    [0014]
    Method according to any one of claims 1 to 13, characterized in that the ultrafiltration membrane has a molecular weight cut of 50 kDa or less, or 30 kDa or less.
    [0015]
    Method according to any one of claims 1 to 14, characterized in that the composition comprises highly concentrated human interleukin-6 anti-receptor monoclonal antibodies, or tocilizumab.
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    同族专利:
    公开号 | 公开日
    JP6363017B2|2018-07-25|
    US9630988B2|2017-04-25|
    KR101993488B1|2019-06-26|
    MX2014002253A|2014-04-30|
    IL231128D0|2014-03-31|
    SG11201400260TA|2014-03-28|
    JP2014526371A|2014-10-06|
    CN103889555B|2018-10-26|
    US20150037319A1|2015-02-05|
    JP2017131886A|2017-08-03|
    EP3235557A1|2017-10-25|
    CA2847302C|2021-02-16|
    NZ621902A|2016-06-24|
    BR112014004828A2|2017-04-04|
    US20170204135A1|2017-07-20|
    JP6363751B2|2018-07-25|
    AU2012303372A1|2014-02-20|
    EP2750784A1|2014-07-09|
    MX359235B|2018-09-20|
    AU2017204395B2|2018-11-08|
    RU2638859C9|2018-01-31|
    RU2014112230A|2015-10-10|
    IL231128A|2019-03-31|
    US20200172569A1|2020-06-04|
    KR20140078642A|2014-06-25|
    RU2638859C2|2017-12-18|
    US10590164B2|2020-03-17|
    AU2017204395A1|2017-07-20|
    CN103889555A|2014-06-25|
    ZA201401184B|2014-12-23|
    WO2013031237A1|2013-03-07|
    CA2847302A1|2013-03-07|
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    法律状态:
    2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2019-10-08| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|
    2020-05-12| B07D| Technical examination (opinion) related to article 229 of industrial property law|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |
    2020-06-09| B07E| Notice of approval relating to section 229 industrial property law|
    2020-06-30| B07A| Technical examination (opinion): publication of technical examination (opinion)|
    2021-01-05| B09A| Decision: intention to grant|
    2021-03-09| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 31/08/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201161530158P| true| 2011-09-01|2011-09-01|
    US61/530,158|2011-09-01|
    PCT/JP2012/005536|WO2013031237A1|2011-09-01|2012-08-31|Method for preparing a composition comprising highly concentrated antibodies by ultrafiltration|
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