METHOD FOR PURIFYING A PROTEIN FROM A SOLUTION

专利摘要:
continuous multistep process for antibody purification. the invention relates to a three-step chromatography process for the small and large scale purification of proteins, specifically monoclonal antibodies, using only four buffer solutions made from a stock solution.
公开号:BR112015027812B1
申请号:R112015027812-4
申请日:2014-05-06
公开日:2021-03-30
发明作者:Didier Duthe；Céline HEMET；Laure Landric-Burtin；Benoit Mothes
申请人:Sanofi；
IPC主号:

专利说明:

TECHNICAL FIELD
[001] The invention relates to a three-stage chromatography process for the purification of small and large-scale proteins, monoclonal antibodies specifically, using four buffer solutions. BACKGROUND
[002] Purification of antibodies can be one of the most expensive aspects of bioproduction. Monoclonal antibodies (mAbs) are generally purified using one of three steps, three resin chromatography processes, using a specific buffer system at each step. This conventional purification process comprises a capture step, followed by an ion exchange step, and concludes with a polishing step, and usually takes 3 to 5 working days (including storage and open phases). In such conventional processes, these three steps are carried out in a sequence of distinct unit operations, which cannot be operated in a continuous way as pH adjustment, the molarity and protein concentration are necessary between each step. Such a conventional purification process is outlined in Figure 5. Therefore, conventional purification processes generally require numerous different buffers, as well as numerous storage units between each discontinued step. These conventional purification processes are therefore prone to contamination, technical failures and human errors. In addition, since an interruption between each step is necessary to concentrate the eluate, adjusting the pH and conductivity and storing the fluid before the next step, and since a step cannot start before the completion of the previous one, such processes Conventional purification methods are particularly long and expensive, as can be seen in Figure 7.
[003] With the increase in cell culture titers and larger cell culture volumes being used for production, further processing is seen as an industry bottleneck. This is particularly relevant for the production of monoclonal antibodies, in which the focus has been shifted away from the batch volume, processing capacity and downstream. In addition, early pre-clinical and clinical studies require larger amounts of antibodies that can be produced more quickly. Therefore, there is a need in the industry for a process, which can be carried out continuously, for the purification of antibodies, and for both a reduction in the time required to obtain batches, risks of contamination, technical failures and human errors and for vertical scalability requirements of the process. SUMMARY OF THE INVENTION
[004] The inventors have discovered a new method for the purification of antibodies, said method comprising a limited number of steps in a continuous mode, using reduced amounts of resins and buffers while still allowing to obtain high yields of purified antibodies with an excellent degree of purity. Purified proteins are therefore suitable for medical applications. Therefore, the method can be used to purify proteins for clinical trials and / or for the commercialization of a pharmaceutical composition comprising the protein. In addition, this method does not require any inter-step adjustment and, therefore, can be performed in a closed system from the collection of proteins to be purified for the final product.
[005] Briefly, this method comprises only three chromatographic steps in a continuous mode: an affinity chromatography, a multimodal or cation exchange resin chromatography, and an anion exchange chromatography (AEX). These three chromatographic steps can be implemented in any order. In addition, it was found that all buffers used during these three chromatography steps can be prepared from the same stock solution. These buffers advantageously comprise Bis Tris, for example, in combination with NaCl, acetic acid, water, and, optionally, NH4 Cl. As there is no need for any buffer changes, the method is easy to perform, and is highly suitable for automation and / or for continuous execution. More particularly, it is possible to use only 4 plugs for the entire process, ensuring compatibility between all stages and allowing to supply chain manufacturing and economy of quality control and reduced storage needs.
[006] The method of the invention also allows to reduce or abolish open phases (that is, steps where the purification system is opened to perform a manual operation, such as preparing the chromatographic column for a new buffer, diluting the sample, or adjusting pH), thus reducing the risk of contamination and giving the possibility to work in a less classified environment. In addition, since each chromatographic step of the method of the present invention can use reusable resins or, surprisingly, can reuse disposable membrane adsorbers, the sequence of three chromatographic steps can be renewed until the desired amount is obtained without human manipulation. In particular, all the chromatographic steps of the method of the invention can be implemented using resins that can be reused at least 100 times or use membrane adsorbents that can be reused at least 50 times. The inventors demonstrated that, in fact, it was possible to use the same disposable membrane adsorber through at least 50 runs without losing stability. The cycle times of the process are thus shortened, the scaling requirements of the process are minimized, and it is possible to reduce operating and storage expenses as the volumes of resins and buffers can be reduced and disposable membrane adsorbents do not need to be stored after a batch. Therefore, the method of the invention allows both cost effective batch production and reduced purification system occupancy time, as can be seen in Figures 10 and 12, in relation to Figures 9 and 11. It is, therefore, suitable scaling up and purification of recombinant proteins from the bank to the industrial scale.
[007] A specific protocol was developed and executed by four different antibodies. In this protocol, the crude protein eluent obtained at the end of the first chromatographic step is passed directly through the second chromatography column, in particular, through the second chromatography column or membrane adsorber, that is, without undergoing any treatment such as pH adjustment. , buffer exchange or dilution, and the protein eluate obtained at the end of the second chromatographic step is also passed directly through the third chromatography column, in particular through the third chromatography column or membrane adsorber, that is, without undergoing any treatment such as pH adjustment, buffer exchange or dilution. This method is outlined in Figure 6. In addition, in this protocol, the protein-containing solution is loaded through the first chromatography column, in particular through the first chromatography column or membrane adsorber, in successive runs (see Example 6), successive runs starting as soon as the previous run is eluted from the first chromatography step, as can be seen in Figure 8. This protocol has the advantage of being extremely fast (about 2 hours for a sequence), leads to an improved performance (more than 90%), better purity and allows to reduce the volumes of buffers and resins used when columns are used, and reduces both buffers and storage facilities used when membrane adsorbents are used. In addition, this process has the advantage of being extremely flexible since the size of the columns used and / or the number of runs can be easily adapted to the amount of proteins to be purified. In addition, it can be completely automated, run in continuous mode, and does not include any open phase. In addition, it was successfully performed by four different antibodies without the need for optimization.
[008] The invention thus provides a method for the purification of a protein from the solution which comprises a first chromatography step comprising the passage of equilibration buffer through a first chromatography column, in particular through a first chromatography column chromatography or membrane adsorber, pass the solution through the first chromatography column, namely through the first chromatography column or membrane adsorbor, pass the equilibration buffer through the first chromatography column, in particular through the first chromatography column or adsorbor membrane, pass the wash buffer through the first chromatography column, in particular through the first chromatography column or membrane adsorbor, pass the equilibration buffer through the first chromatography column, in particular through the first chromatography column or adsorbor membrane, and elute a crude protein eluent to from the first chromatography column, namely from the first chromatography column or membrane adsorber, use a first elution buffer; a second chromatography step comprising the equilibration buffer through a second chromatography column, in particular on a second chromatography column or membrane adsorber, passing the crude protein eluent through the second chromatography column, in particular, through the second chromatography column or membrane adsorber, optionally, pass the equilibration buffer through the second chromatography column, in particular, through the second chromatography column or membrane adsorbor, and elute a protein eluate from the second chromatography column , in particular from the second chromatography column or membrane adsorber, using a second elution buffer; and a third chromatography step comprising the passage of equilibration buffer through a third column of chromatography, in particular on a third column of chromatography or membrane adsorbent, passing the protein eluate through the third column of chromatography, namely through third chromatography column or membrane adsorber, in the pass-through flow mode optionally pass the wash buffer through the third chromatography column, in particular through the third chromatography column or membrane adsorbor, and recover the purified protein from of the passage flow of the third chromatography column, namely the third chromatography column or membrane adsorber.
[009] The invention also provides a method for the purification of a protein from a solution comprising a first chromatography step comprising the passage of equilibration buffer through a first chromatography column, in particular through a first column chromatography or membrane adsorber, pass a part of the solution through the first chromatography column, in particular, through the first chromatography column or membrane adsorbor, pass the equilibration buffer through the first chromatography column, in particular through the first chromatography column or membrane adsorber, pass the wash buffer through the first chromatography column, in particular through the first chromatography column or membrane adsorbor, pass the equilibration buffer through the first chromatography column, in particular through the first chromatography column or membrane adsorber, elute an eluent of crude protein from the first chromatography column, namely from the first chromatography column or membrane adsorber, use a first elution buffer, and optionally pass the sanitation buffer through the first chromatography column, in particular through the first chromatography column or membrane adsorber; a second chromatography step comprising the passage of equilibration buffer through a second chromatography column, in particular on a second chromatography column or membrane adsorbent, passing the crude protein eluent through the second chromatography column, in particular, through the second column of chromatography or membrane adsorber, optionally, pass the equilibration buffer through the second column of chromatography, in particular, through the second column of chromatography or membrane adsorbor, elute a protein eluate from the second column of chromatography, in particular from the second chromatography column or membrane adsorber, use a second elution buffer, and optionally pass sanitation buffer through the second chromatography column, in particular, through the second chromatography column or membrane adsorbor; a third chromatography step comprising the passage of equilibration buffer through a third chromatography column, in particular on a third chromatography column or membrane adsorbent, passing the protein eluate through the third chromatography column, namely through the third chromatography column or membrane adsorber, in flow-through mode, optionally, pass the wash buffer through the third chromatography column, in particular through the third chromatography column or membrane adsorber, recover purified protein from the flow passing the third chromatography column, in particular the third chromatography column or membrane adsorber, and optionally passing sanitation buffer through the third chromatography column, in particular through the third chromatography column or membrane adsorbor; successively renew the first, second and third chromatography steps with another part of the solution until all the solution is used, and collect the purified proteins recovered at the end of each third chromatography step.
[010] In one embodiment of the invention, each of the plugs comprises Bis Tris. In another embodiment, each buffer comprises Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl. The use of Bis Tris buffers in the method of the invention is particularly important because it makes it possible to avoid adjusting the pH between the three chromatographic steps and thus, executing the method in a closed system, from the first to the last step.
[011] In one embodiment, one of the chromatography columns is a Protein A matrix. In one embodiment, one of the chromatography columns is a multimodal resin or cation exchange chromatography column. In one embodiment, one of the chromatography columns is an anion exchange chromatography column.
[012] In a particular embodiment, the method of the invention comprises a Protein A chromatography column, a multimodal or cation exchange resin chromatography column and an anion exchange chromatography column, said matrices being used in any order during the three chromatographic steps.
[013] In one embodiment of the invention, the first chromatography column is a Protein A matrix, the second chromatography column is a multimodal resin or cation exchange chromatography column and the third chromatography column is a chromatography column anion exchange.
[014] In one embodiment of the invention, each chromatography column is a chromatography column. In a particular embodiment of this modality, the first chromatography column is a Protein A column, the second chromatography column is a multimodal resin chromatography column and the third chromatography column is an anion exchange chromatography column.
[015] In another embodiment of the invention, each chromatography column is a chromatography membrane adsorber. In a particular embodiment of this modality, the first chromatography column is a Protein A membrane adsorber, the second chromatography column is a cation exchange membrane adsorber and the third chromatography column is an anion exchange membrane adsorbent.
[016] In one embodiment of the invention, the protein to be purified is an antibody. In another embodiment, the antibody is a monoclonal antibody.
[017] In one embodiment of the invention, the method additionally comprises a nanofiltration step after step (c) and / or an ultrafiltration and diafiltration step after the nanofiltration step. In another embodiment of the invention, the method additionally comprises a low pH inactivation step after step (c), after the nanofiltration step and / or after the ultrafiltration and diafiltration step. In one embodiment of the invention, the method comprises, prior to step (a), a cell culture step in a liquid culture medium, preferably in a bioreactor, to provide a liquid culture medium containing the protein. The cultured cells can be bacterial, fungal or mammalian cells.
[018] The invention, therefore, also provides an integrated process for the production of a purified protein from a liquid culture medium.
[019] In certain embodiments of the invention, the first elution buffer comprises 20 mM Bis Tris, and 20 mM NaCl, adjusted to pH 3.7 with acetic acid; the second elution buffer comprises 20 mM Bis Tris, 45 mM NaCl and 25 mM NH4Cl adjusted to pH 7.25 with acetic acid or comprises 20 mM Bis Tris, 80 mM NaCl and 25 mM NH4Cl adjusted to pH 6.2 with acetic acid; the equilibration buffer comprises 20 mM Bis Tris, and 20 mM NaCl, adjusted to pH 7.4 with acetic acid; and the wash buffer comprises 20 mM Bis Tris, and 1 M NaCl adjusted to pH 7.4 with acetic acid. In other embodiments of the invention, the sanitation buffer comprises 0.1 N sodium hydroxide.
[020] The invention provides a kit comprising a multimodal or cation exchange resin chromatography column, a protein A matrix and / or an anion exchange chromatography column; and at least one buffer comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl. In some embodiments, the kit is used for the purification of a protein from the solution using a method of the invention.
[021] In one embodiment, the kit comprises a multimodal resin chromatography column, a protein column and / or an anion exchange chromatography column; and at least one buffer comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl.
[022] In another embodiment, the kit comprises a cation exchange membrane adsorber, a Protein A membrane adsorber and / or an anion exchange membrane adsorber; and at least one buffer comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl.
[023] The invention also provides a kit comprising a multimodal or cation exchange resin chromatography column, a protein A matrix and / or an anion exchange chromatography column; and instructions for preparing at least one buffer comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl. In some embodiments, the kit is used for the purification of a protein from the solution using a method of the invention.
[024] In one embodiment, the kit comprises a multimodal resin chromatography column, a Protein A column and / or an anion exchange chromatography; and instructions for preparing at least one buffer comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl.
[025] In another embodiment, the kit comprises a cation exchange membrane adsorber, a Protein A membrane adsorber and / or an anion exchange membrane adsorber; and instructions for preparing at least one buffer comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl.
[026] The invention further provides a method for the preparation of equilibration buffer comprising creating a 100 L solution with a final concentration of 20 mM Bis Tris and 20 mM NaCl; adjusting the pH of the solution to 7.4 with acetic acid; and collecting 25 L of the solution. The invention also provides a method for the preparation of wash buffer which comprises collecting 25 L of the remaining 75 L of solution from the preparation of the equilibration buffer and adding 1 M eq NaCl to these 25 L of solution. The invention further provides a method for the preparation of an elution buffer which comprises collecting 25 L of the remaining 50 L of solution from the preparation of the equilibration buffer and adjusting the pH of these 25 L of solution to 3.7 with acid acetic. The invention further provides a method for the preparation of an elution buffer comprising the addition of 45 mM Naq eq and 25 mM NH4Cl eq to the remaining 25 L of solution from the preparation of the equilibration buffer and adjusting the pH of these 25 L to 7.25 with acetic acid. The buffers prepared by the methods described herein can be used for the purification of a protein from the solution using a method of the invention.
[027] Also provided herein are isolated proteins, pharmaceutical agents and pharmaceutical compositions obtained by any of the methods described herein.
[028] These and other features and advantages of the described purification method will be more fully understood from the following detailed description taken in conjunction with the appended claims. It is noted that the scope of the claims is defined by the recitations in them and not by the specific discussion of characteristics and advantages established in the description.
[029] In the context of the invention, the terms "comprising", "having", "including" and "containing" are to be interpreted as open terms (ie, which means "including, but not limited to,"), unless otherwise indicated. In addition, the term "comprising" encompasses "consisting" (for example, a composition "comprising" X may consist exclusively of X or may include something additional, for example, X + Y). BRIEF DESCRIPTION OF THE DRAWINGS
[030] The following detailed description of the described purification method modalities can best be understood when read in conjunction with the following drawings.
[031] Figure 1 shows a schematic representation of the protocol used to formulate the purification method buffers described in Examples 2 to 7.
[032] Figure 2 shows graphs representing the sweet spots for the second elution buffer for three concentrations of NH4CI.
[033] Figure 3 shows the graphs that represent the analysis of HMW trend, yield, HCP and DNA in each of the 15 runs of Example 5.
[034] Figure 4 shows graphs that represent the trend analysis of HMW, LMW, HCP and DNA in each of the 50 runs of Example 8.
[035] Figure 5 shows a schematic representation of the different steps of a conventional process for the purification of proteins. BH: mass collection; EqB # 1: first balance buffer; WB # 1: first wash buffer, EIB # 1: first elution buffer; EqB # 2: second balance buffer; WB # 2: second wash buffer, EIB # 2: second elution buffer; EqB # 3: third balance buffer; WB # 3: third wash buffer; chrom1: first chromatographic step; chrom2: second chromatographic step; chrom3: third chromatographic stage; adj. pH: pH adjustment; conc. adjust .: concentration adjustment; adj. cond .: conductivity adjustment; samp .: sample; Stor .: storage; filt .: filtration; Nanofilt .: nanofiltration; TFF: tangential flow filtration.
[036] Figure 6 shows a schematic representation of the different steps of the method of the invention. BH: mass collection; EqB # 1: first balance buffer; WB # 1: first wash buffer, EIB # 1: first elution buffer; EIB # 2: second elution buffer; chrom1: first chromatographic step; chrom2: second chromatographic step; chrom3: third chromatographic stage; samp .: sampling; Stor .: storage; Nanof .: nanofiltration; TFF: tangential flow filtration.
[037] Figure 7 shows a schematic representation of the different steps of a conventional process for the purification of proteins including several runs or cycles. Clar .: clarification; EqB # 1: first balance buffer; WB # 1: first wash buffer, EIB # 1: first elution buffer; EqB # 2: second balance buffer; WB # 2: second wash buffer, BEI # 2: second elution buffer; EqB # 3: third balance buffer; WB # 3: third wash buffer; chrom1: first chromatographic step; chrom2: second chromatographic step; chrom3: third chromatographic stage; Nanof .: nanofiltration; UF / DF: ultrafiltration / diafiltration.
[038] Figure 8 shows a schematic representation of the different steps of the method of the invention, including several executions or cycles. Clar .: clarification; EqB # 1: first balance buffer; WB # 1: first wash buffer, EIB # 1: first elution buffer; EIB # 2: second elution buffer; chrom1: first chromatographic step; chrom2: second chromatographic step; chrom3: third chromatographic stage; Nanof .: nanofiltration; UF / DF: ultrafiltration / diafiltration.
[039] Figure 9 shows a schematic view of a timeline of the different steps in a conventional protein purification process including several runs or cycles. The first row of the table shows the time in hours. Clar .: clarification; chrom1: first chromatographic step; chrom2: second chromatographic step; chrom3: third chromatographic stage; Low pH inact .: low pH inactivation; Nanof .: nanofiltration; UF / DF: ultrafiltration / diafiltration. The circle represents the moment when the process is completed.
[040] Figure 10 shows a schematic of a timeline of the different steps of the method of the invention, including several runs or cycles. Clar .: clarification; chrom1: first chromatographic step; chrom2: second chromatographic step; chrom3: third chromatographic stage; Nanof .: nanofiltration; UF / DF: ultrafiltration / diafiltration. The circle represents the moment when the process is completed.
[041] Figure 11 shows a schematic of a timeline of the different steps of a conventional process for the purification of proteins including several runs or cycles. The first row of the table shows the time in hours. Clar .: clarification; chrom1: first chromatographic step; chrom2: second chromatographic step; chrom3: third chromatographic stage; Low pH inact .: low pH inactivation; Nanof .: nanofiltration; UF / DF: ultrafiltration / diafiltration. The level of estimation of the productivity of the process is indicated.
[042] Figure 12 shows a schematic of a timeline of the different steps of the method of the invention, including several runs or cycles. Clar .: clarification; chrl: first chromatographic step; chr2: second chromatographic step; chrl3: third chromatographic step; Nanof .: nanofiltration; UF / DF: ultrafiltration / diafiltration. The level of estimation of the productivity of the process is indicated. DETAILED DESCRIPTION OF MODALITIES AND ASPECTS
[043] Based on the availability of mixed-mode resins (also called multimodal resins) and chromatography membrane adsorbents, the inventors developed a new purification process using only three chromatography steps. In other words, the method comprises only three steps involving passing through a chromatography column.
[044] The invention relates to a method for the purification of a protein from the solution comprising or consisting of: (a) a first chromatography step comprising: - passing said solution through a first chromatography column; - eluting a crude protein eluent from the first chromatography column using a first elution buffer; (b) a second chromatography step comprising: - passing the crude protein eluent obtained at the end of step (a) through a second chromatography column; - eluting a protein eluate from the second chromatography column using a second elution buffer; and (c) a third chromatography step comprising: - passing the protein eluate obtained at the end of step (b) through a third chromatography column in the flow-through mode; - recovering purified protein from the passage flow of the third chromatography column; wherein each of the plugs comprises Bis Tris.
[045] More specifically, each of the first two chromatography steps above may comprise or consist of: - passing the equilibration buffer through the chromatography column; - passing the crude protein solution or eluent through the chromatography column (as mentioned above); - pass the equilibration buffer through the chromatography column; - optionally pass wash buffer through the chromatography column; - optionally pass equilibration buffer through the chromatography column; - eluting the crude protein eluent or the protein eluate from the chromatography column using an elution buffer (as mentioned above), each buffer comprising Bis Tris.
[046] The invention also relates to a method for the purification of a protein from the solution comprising or consisting of: (a) a first chromatography step comprising: - passing a part of said solution through a first chromatography column ; - eluting a crude protein eluent from the first chromatography column using a first elution buffer, and - optionally, pass the sanitation buffer through the first chromatography column; (b) a second chromatography step comprising: - passing the crude protein eluent obtained at the end of step (a) through a second chromatography column, - eluting a protein eluate from the second chromatography column using a second buffer elution, and - optionally, pass the sanitation buffer through the second chromatography column; (c) a third chromatography step comprising: - passing the protein eluate obtained at the end of step (b) through a third chromatography column in the flow-through mode, - recovering purified protein from the flow-through of the third chromatography column, and - optionally pass sanitation buffer through the third chromatography column; successively renew steps (a), (b) and (c) with another part of the solution until all the solution is used, and collect the purified proteins recovered at the end of each step (c); wherein each of the equilibration, wash and elution buffers comprises Bis Tris.
[047] In the context of the invention, the term "chromatography column" refers to any type of means for absorbing particles, resin or other solid phase, such as a membrane, which, in a purification process, acts as the absorbent to separate the molecule to be purified from other molecules present in a mixture. The matrix, in particular, which consists of resin matrices, can be in the form of columns, or in the form of membrane adsorbents.
[048] In the context of the invention, a "membrane adsorber" refers to a flat sheet of acrylic polymer, having ionic groups and comprising linked functional groups, such as affinity groups and ion exchange groups. One of the differences between the resin and the membrane is the flow distribution: by diffusion to the resin and by convection in membranes.
[049] In one embodiment of the method of the invention, the first, second and third chromatography columns are chromatography columns. In another embodiment of the method of the invention, the first, second and third chromatography columns are chromatography membrane adsorbers.
[050] Therefore, in one embodiment, the invention relates to a method for the purification of a protein from the solution comprising or consisting of: (a) a first chromatography step comprising: - passing said solution through a first chromatography column; - Elute a crude protein eluent from the first chromatography column, using a first elution buffer; (b) a second chromatography step comprising: - passing the crude protein eluent obtained at the end of step (a) through a second chromatography column; - eluting a protein eluate from the second chromatography column with a second elution buffer; and (c) a third chromatography step comprising: - passing the protein eluate obtained at the end of step (b) through a third chromatography column in the flow-through mode; - recovering purified protein from the passage flow of the third chromatography column; wherein each of the plugs comprises Bis Tris.
[051] More specifically, each of the first two chromatography steps above may comprise or consist of: - passing the equilibration buffer through the chromatography column; - passing the crude protein solution or eluent through the chromatography column (as mentioned above); - pass equilibration buffer through the chromatography column; - optionally pass the wash buffer along the chromatography column; - optionally pass equilibration buffer through the chromatography column; - eluting the crude protein eluent or protein eluate from the chromatography column using an elution buffer (as mentioned above), each buffer comprising Bis Tris.
[052] The invention also relates to a method for the purification of a protein from the solution comprising or consisting of: (a) a first chromatography step comprising: - passing a part of said solution through a first chromatography column ; - eluting a crude protein eluent from the first chromatography column, using a first elution buffer, and - optionally, pass the sanitation buffer through the first chromatography column; (b) a second chromatography step comprising: - passing the crude protein eluent obtained at the end of step (a) through a second chromatography column, - eluting a protein eluate from the second chromatography column with a second buffer elution, and - optionally, pass the sanitation buffer through the second chromatography column; (c) a third chromatography step comprising: - passing the protein eluate obtained at the end of step (b) through a third chromatography column in the flow-through mode, - recovering purified protein from the flow-through of the third chromatography column, and - optionally pass sanitation buffer through the third chromatography column; successively renew steps (a), (b) and (c) with another part of the solution until all the solution is used, and collect the purified proteins recovered at the end of each step (c); wherein each of the equilibration, wash and elution buffers comprises Bis Tris.
[053] In another embodiment, the invention relates to a method for the purification of a protein from the solution comprising or consisting of: (a) a first chromatography step comprising: - passing said solution through a first chromatography membrane adsorber; - eluting a crude protein eluent from the first chromatography membrane adsorber using a first elution buffer; (b) a second chromatography step comprising: - passing the crude protein eluent obtained at the end of step (a) through a second chromatography membrane adsorbent; - eluting a protein eluate from the second chromatography membrane adsorber using a second elution buffer; and (c) a third chromatography step comprising: - passing the protein eluate obtained at the end of step (b) through a third chromatography membrane adsorber in the flow-through mode; - recovering purified protein from the passage flow of the third chromatography membrane adsorber; wherein each of the plugs comprises Bis Tris.
[054] More specifically, each of the first two chromatography steps above may comprise or consist of: - passing equilibration buffer through the chromatography membrane adsorber; - passing the crude protein solution or eluent through the chromatography membrane adsorber (as mentioned above); - pass the Balance Buffer through the chromatography membrane adsorber; - optionally pass wash buffer through the chromatography membrane adsorber; - optionally pass equilibration buffer through the chromatography membrane adsorber; - eluting the crude protein eluent or protein eluate from the chromatography membrane adsorber using an elution buffer (as mentioned above), each buffer comprising Bis Tris.
[055] The invention also relates to a method for the purification of a protein from the solution comprising or consisting of: (a) a first chromatography step comprising: - passing a part of said solution through a first adsorber of chromatography membrane; - eluting a crude protein eluent from the first chromatography membrane adsorber using a first elution buffer, and - optionally, pass the sanitation buffer through the first chromatography membrane adsorber; (b) a second chromatography step comprising: 1) passing the crude protein eluent obtained at the end of step (a) through a second chromatography membrane adsorbent, 2) eluting a protein eluate from the second membrane adsorbent chromatography using a second elution buffer, and 3) optionally, pass the sanitation buffer in relation to the second chromatography membrane adsorber; (c) a third chromatography step comprising: - passing the protein eluate obtained at the end of step (b) through a third chromatography membrane adsorber in flow-through mode, - recovering the purified protein from the flow of passage of the third chromatography membrane adsorber, and - optionally pass sanitation buffer through the third chromatography membrane adsorber; successively renew steps (a), (b) and (c) with another part of the solution until all the solution is used, and collect the purified proteins recovered at the end of each step (c); wherein each of the equilibration, wash and elution buffers comprises Bis Tris.
[056] As indicated above, the aforementioned method of the invention comprises only three chromatography steps. Although the method according to the invention comprises only three chromatography steps, it allows obtaining purified proteins that are suitable for pharmaceutical purposes and, in particular, for administration to humans.
[057] In addition to the absence of human manipulation in the purification process (and consequent reduction in the total time required to complete the purification process), the described process reduces the amount of buffers and resins used for purification. In addition, the main buffers comprise the same components (ie, Bis Tris, NaCl, acetic acid, water and, optionally, NH4Cl), which greatly facilitates the preparation of the buffer. The disclosed purification method also simplifies the purification of mAb, improves total yield, and reduces raw materials, storage facilities, cost of goods and process time, in addition to allowing the purification of a variety of mAbs.
[058] In contrast to conventional protein purification methods, as indicated above, the method disclosed here uses four or five buffers: a balance buffer, a wash buffer, two elution buffers, and optionally a sanitation buffer. The four main buffers used in the disclosed method are made with the same matrix of compounds, from a stock solution, which greatly facilitates the preparation of the buffer.
[059] As used herein, "buffers according to the invention" refer to buffers that comprise Bis Tris. Bis Tris is a compound well known to the person skilled in the art, the name IUPAC which is 2- [bi (2-hydroxyethyl) amino] -2- (hydroxymethyl) propane-1,3-diol, and the CAS Number which is 6976-37-0. Such buffers according to the invention may correspond to an equilibration buffer, a wash buffer, and / or an elution buffer.
[060] More specifically, such buffers according to the invention may comprise or consist of various concentrations of the same chemicals (one being Bis Tris). In a specific embodiment, the buffers comprise or consist of Bis Tris, acetic acid and water. In a more specific embodiment, the buffers comprise or consist of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl. In other words, such buffers comprise or consist of various concentrations of Bis Tris, acetic acid, NaCl, water and, optionally, NH4Cl.
[061] The elution buffer may, for example, comprise or consist of 15 to 25 mm (for example, 20 mM) Bis Tris, and 15 to 25 mm (for example, 20 mM) NaCl, adjusted to a pH between 3 and 4 (eg 3.7) with acetic acid. Such an elution buffer is especially suitable for use with an affinity chromatography column, in particular, with an affinity chromatography column or membrane adsorber, such as a protein A matrix, in particular a protein A column or a protein membrane adsorber.
[062] The elution buffer may further comprise or consist of 15 to 25 mm (for example, 20 mM) of Bis Tris, 40 to 50 mM (for example, 45 mM) of NaCl, and 20 to 30 mM (for example , 25 mM) NH4Cl, adjusted to a pH between 7 and 8 (for example, 7.25) with acetic acid. Such an elution buffer is especially for use with a multimodal resin chromatography column, in particular, with a multimodal resin chromatography column, such as, for example, Capto MMC.
[063] The elution buffer may further comprise or consist of 15 to 25 mM (for example, 20 mM) of Bis Tris, 50 to 150 mM (for example 80 mM) of NaCl, and 20 to 30 mM (for example, 25 mM) NH4Cl, adjusted to a pH between 6 and 7 (for example, 6.2) with acetic acid. Such an elution buffer is especially for use with a cation exchange chromatography column, in particular, with a cation exchange membrane adsorber.
[064] The balance buffer may comprise or consist of 15 to 25 mM (for example, 20 mM) of Bis Tris, and 15 to 25 mM (for example 20 mM) of NaCl, adjusted to a pH between 7 and 8 (eg 7.4) with acetic acid.
[065] The wash buffer may comprise or consist of 15 to 25 mM (eg, 20 mM) Bis Bis, and 0.9 to 1.1 M (eg 1 M) NaCl adjusted to a pH between 7 and 8 (for example 7.4) with acetic acid.
[066] More specifically, a balance buffer for use in the disclosed method contains 20 mM Bis Tris and 20 mM NaCl, adjusted to pH 7.4 with 2 mM acetic acid. A wash buffer for use in the disclosed method contains 20 mM Tris Bis and 1 M NaCl, adjusted to pH 7.4 with 2 mM acetic acid. A first elution buffer for use in the disclosed method contains 20 mM Bis Tris and 20 mM NaCl, adjusted to pH 3.7 with 275 mM acetic acid. A second elution buffer for use in the disclosed method contains 20 mM Bis Tris, 45 mM NaCl, and 25 mM NH4Cl adjusted to pH 7.25 with 5 mM acetic acid, in particular for use with a chromatography column of multimodal resin, or contains 20 mM Bis Tris, 80 mM NaCl, and 25 mM NH4Cl adjusted to pH 6.2 with 5 mM acetic acid, in particular for use with a cation exchange membrane adsorbent.
[067] Advantages of previous buffer formulations include the ability of an mAb product to pass through the three chromatography columns, in particular, the three chromatography columns or the three chromatography membrane adsorbers, used in the method described to a greater degree compatibility, while minimizing unwanted interactions, limiting pH and drop in conductivity, and promoting higher yields against traditional purification methods. In addition to the use of a reduced number of buffers, another aspect of the disclosed method is the use of a Bis-Tris buffer. The use of such a buffer avoids pH adjustment between the three chromatographic steps and, therefore, allows the method to be run in a closed system from collection to the last purification step.
[068] The sanitation buffer optionally used in the context of the invention can comprise or consist of 0.05 N and 0.15 N (for example, 0.1 N) NaOH. Such a sanitation buffer is especially suitable for use with an affinity chromatography column, in particular, with an affinity chromatography column, such as Protein A column or with an affinity chromatography membrane adsorber such as Protein A Sartobind, with a multimodal resin chromatography column or cation exchange, namely with a multimodal resin chromatography column, such as Capto MMC, or with a cation exchange chromatography membrane adsorber, as S Sartobind membrane adsorber, and / or with an anion exchange chromatography column, in particular with an anion exchange chromatography column, such as BioPro Q75, or with an anion exchange chromatography membrane adsorber, such as Q Sartobind membrane adsorber.
[069] The terms "polypeptide" or "protein", as used herein, refer to: 5) molecules having the sequence of native proteins, which is: a) proteins produced by naturally occurring and specifically non-recombinant cells, or b) by genetic engineering cells, or recombinants, or 6) molecules that differ from the sequence of native proteins by deletions, additions to, and / or substitutions of one or more amino acids and / or by at least one post-modification translation (eg glycosylation).
[070] The molecules mentioned in paragraph 1) above can be called native proteins. The molecules mentioned in paragraph 2) above are unnatural proteins.
[071] In certain respects, the protein to be purified is an antibody.
[072] The term "antibody" as used herein refers to an intact antibody, or a binding fragment thereof, that competes with the intact antibody for specific binding. Binding fragments include, but are not limited to, F (ab), F (ab '), F (ab') 2, Fv and single chain antibodies. The term "heavy chain" includes any polypeptide having an immunoglobulin sequence of the variable region sufficient to confer specificity for an antigen.
[073] The term "heavy chain", as used herein, encompasses a full-length heavy chain and fragments thereof. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino terminal end of the polypeptide, and the CH3 domain is at the carboxyl terminal.
[074] The term "light chain", as used herein, encompasses a full-length light chain and its fragments. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. Like the heavy chain, the domain of the light chain variable region is at the amino terminus of the polypeptide. The term "light chain", as used herein, includes any polypeptide having an immunoglobulin sequence of the variable region sufficient to confer specificity for an antigen.
[075] Structural units of naturally occurring antibodies typically comprise a tetramer. Each such tetramer is usually composed of two identical pairs of polypeptide chains, each pair having a total light chain length (typically having a molecular weight of about 25 kDa) and a full length heavy chain (typically having a weight molecular weight of about 50-70 kDa). The amino-terminal portion of each heavy and light chain typically includes a variable region of about 100 to 110 or more amino acids that is typically responsible for antigen recognition. The carboxy-terminal portion of each chain normally defines a constant region responsible for the effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are classified as mu, typically delta, gamma, alpha, or epsilon, and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, lgM1 and lgM2. IgA is similarly subdivided into subclasses that include, but are not limited to, lgA1 and lgA2. Within the full-length light and heavy chains, the variable and constant regions are typically joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. .
[076] The variable regions of each light / heavy chain pair normally form the antigen binding site. The variable regions typically exhibit the same general structure as relatively conserved structural regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs of the two chains in each pair are typically aligned with the structural regions, which can allow binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. The assignment of amino acids to each domain is typically according to the definitions in Kabat et al., 1991, Sequence of Proteins of Immunological Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242 USA. A bispecific or bifunctional antibody is usually an artificial hybrid antibody, having two different heavy chain / light chain pairs and two different binding sites.
[077] An AF (ab) fragment consists of a light chain and CH1 and variable regions of a heavy chain. The heavy chain of an F (ab) molecule may not form a disulfide bond with another heavy chain molecule. Fragment AF (ab ') contains a light chain and a heavy chain that contains more of the constant region, between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between two heavy chains to form an F ( ab ') 2. The Fv region comprises the variable regions of both the heavy and light chains, but the constant regions are missing. Single chain antibodies are Fv molecules in which the variable regions of the heavy and light chains have been linked by a flexible linker to form a single polypeptide chain, which forms an antigen-binding region. A divalent antibody other than a "multispecific" or "multifunctional" antibody, in certain embodiments, is understood to include binding sites that have identical antigen specificity.
[078] Monoclonal antibodies (mAbs) that can be purified by the described process can be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example, the standard somatic cell hybridization technique well known in the art. Although somatic cell hybridization procedures are preferred, in principle, other techniques can be employed for the production of monoclonal antibodies, for example, viral or oncogenic transformation of B-lymphocytes. The monoclonal antibody can, for example, correspond to a murine, a chimeric antibody, a humanized antibody or a fully human one.
[079] In a specific embodiment, the antibody purified by the method of the invention is a monoclonal antibody selected from the group consisting of an antibody that specifically binds to the protofibrillar form of the human β-amyloid protein (for example, a humanized antibody) , an antibody that specifically binds to bacterial surface polysaccharide poly-N-acetyl glucosamine (PNAG) (for example, a fully human antibody), an antibody that specifically binds to cell adhesion molecule related to carcinoembryonic antigen 5 ( CEACAM5) and an antibody that specifically binds to CD38 transmembrane glycoprotein (for example, a humanized antibody).
[080] Non-limiting examples of antibodies that can be purified by the method of the invention also comprise: panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alemtuzumab, aliromacumab, abiromabuma, altirumabuma, altirumama , atinumab, tocilizumab, basilizimab, bectumomab, belimumab, bevacizumab, biciromab, canakinumab, cetuximab, daclizumab, densumab, eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab, etertumaxomab, etertumaxomab, etertumaxomab, etertumaxomab, ettumabumab , rituximab, tocilizumab, trastuzumab, dupilumab, sarilumab or fresolimumab.
[081] In certain respects, the protein to be purified is an enzyme.
[082] Non-limiting examples of enzymes that can be purified by the method of the invention include α-glucosidase acid, α-L-iduronidase, iduronate sulfatase, heparan N-sulfatase, galactose-6-sulfatase, β-galactosidase, β-glucoronidase acid , N-acetylglycosamine-1-phosphotransferase, α-N-acetylgalactosaminidase (α-galactosidase B), acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, β-glucosidase, galactosylceramidase, α-galactosidase A, β-galases galactosidase, neuraminidase, hexosaminidase A or hexosaminidase B.
[083] Other non-limiting examples of proteins that can be purified by the method of the invention include human erythropoietin, tumor necrosis factor (e.g., TNF-α, TNF-β or TNF-K), alpha interferon or interferon beta.
[084] The solution containing the protein to be purified can be a culture medium, preferably a clarified culture medium. The solution containing the protein to be purified is, for example, a culture medium obtained in a perfusion bioreactor, or batch fed bioreactor.
[085] Examples of perfusion bioreactors or batch fed bioreactors are disclosed in provisional U.S. patent application 61 / 775,060 (incorporated herein by reference in its entirety).
[086] The term "clarified culture medium" means a liquid culture medium obtained from, a bacterial cell or mammalian yeast culture that is substantially free (for example, at least 80%, 85%, 90% , 92%, 94%, 96%, 98%, or 99% free) of mammals, bacteria or yeast cells.
[087] The term "protein recovery" as used herein refers to the collection of a protein after using the described purification method. The disclosed purification method can be achieved using a variety of standard protein chromatographic techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and gel chromatography, multimodal resin.
[088] In certain embodiments of the disclosed method, the first chromatography column is a matrix protein. In particular embodiments of the disclosed method, the first chromatography column is a protein column. In other particular embodiments of the disclosed method, the first chromatography column is a Protein A membrane adsorber. The protein A matrix, in particular, the Protein A column or Protein A membrane adsorber, through affinity functions between the resin ligand and the protein, resulting in high impurity removal efficiency. Another advantage of using a protein A matrix, in particular, the use of a Protein A column or a Protein A membrane adsorber, in the method described is that the mAbs have universal affinity for Protein A. In one embodiment of the method described, the Protein A column is MabSelect Sure resin (GE Healthcare). In another modality of the disclosed method, Protein A Column is Absolute Hihg Cap (Novasep). In one embodiment of the method described, the protein is a membrane-adsorbent adsorber of membrane Sartobind Protein A (Sartorius).
[089] In additional modalities of the described process, the second chromatography column is a chromatography column of multimodal resin (mixed mode) or cation exchange resin. In particular embodiments of the method described, the second chromatography column is a multimodal resin chromatography column (mixed mode). The multimodal resin interacts with the protein of interest through several mechanisms with mAb interactions: ionic, hydrophobic and hydrogen. More specifically, a multimodal resin chromatography column, mAb: ionic interaction is a mAb: cationic interaction, as opposed to mAb: anionic interactions that occur in a classic anion exchange chromatography (AEX) column.
[090] In a specific modality of the described process, the multimodal resin is Capto MMC resin (GE Healthcare). Capto MMC is a multimodal cation exchanger with a highly cross-linked agarose-based matrix. The characteristics of Capto MMC are summarized below (see GE Healthcare Life Sciences, data file 11- 0035-4 AA).


[091] In other particular embodiments of the method described, the second column of chromatography is a cation exchange membrane adsorber.
[092] In another specific modality of the described process, the cation exchange membrane adsorber is S Sartobind membrane adsorber (Sartorius). In another specific modality of the described process, the cation exchange membrane adsorber is NatriPur HD-C membrane adsorber (Natrix).
[093] In additional embodiments of the described process, the third chromatography column is an anion exchange chromatography column. In particular embodiments of the method described, the third chromatography column is an anion exchange chromatography column. In other particular embodiments of the disclosed method, the third column of chromatography is an anion exchange membrane adsorber. The positively charged organic portion covalently cross-linked to an inert polymeric support of the anion exchange matrix, in particular the anion exchange resin, interacts with the protein of interest through mAb: anionic interactions. In an embodiment of the method described, the anion exchange chromatography column is BioPro Q75 (YMC). The characteristics of the BioPro Q75 are summarized below (see data sheet YMC-BioPro Q75 and S75).

[094] In another modality of the disclosed method, the anion exchange membrane adsorber is the Sartobind Q membrane adsorber (Sartorius). In another modality of the disclosed method, the anion exchange membrane adsorber is Sartobind STIC membrane adsorber (Sartorius). In another modality of the disclosed method, the anion exchange membrane adsorber is HD-Q membrane adsorber (Natrix).
[095] In one embodiment, the method according to the invention does not comprise adjusting the pH of the crude protein eluent and / or the protein eluate at the end of the first chromatographic step and / or at the end of the second chromatographic step.
[096] In a particular embodiment, the crude protein eluent obtained at the end of the first chromatographic step is passed directly through the second chromatography column, in particular, through the second chromatography column or membrane adsorber. More specifically, no treatment (such as pH adjustment, buffer exchange or dilution) is then carried out between the two steps. In such a method, the multimodal resin chromatography column can, for example, correspond to a Capto MMC column. In such a method, the cation exchange membrane adsorber, for example, corresponds to a Sartobind S membrane adsorber. In addition, in a particular embodiment, the protein eluate obtained at the end of the second chromatographic step is directly transmitted through the third column chromatography, namely through the third column of chromatography or membrane adsorber. More specifically, no treatment (such as pH adjustment, buffer exchange or dilution) is then carried out between the two steps. In such a method, the multimodal resin chromatography column can, for example, correspond to a Capto MMC column and / or the anion exchange chromatography column, for example, can correspond to a BioPro Q75 column. A specific example of this method is described in Example 4. In such a method, the cation exchange membrane adsorbent can, for example, correspond to a Sartobind S membrane adsorbent and / or anion exchange chromatography membrane adsorber can, for example, example, correspond to a Sartobind Q membrane adsorber. A specific example of this method is described in Example 8.
[097] In such a method, inter-step treatments that require manual intervention and opening of the purification system (for example, dilution in an inactivation vial, post-inactivity filtration and pH adjustment in a vial in group A protein) are totally absent.
[098] A chromatography column used in methods of the present invention can be a reduced biological load chromatography column (for example, a gamma-irradiated chromatography column). Examples of a reduced biological load chromatography column are disclosed in provisional US patent application 61 / 928,906, which is hereby incorporated by reference in its entirety.
[099] The method of the invention can, therefore, be carried out in an MCCS which comprises a first, second and third chromatography columns.
[100] The term "multicolumn chromatography system" or "MCCS" means a system of a total of two or more switched or interconnected chromatography columns and / or chromatographic membranes. A non-limiting example of a multicolumn chromatography system is a periodic countercurrent chromatography system (PCCS) containing a total of two or more switching or interconnected chromatography columns and / or chromatographic membranes. Other examples of multi-column chromatography systems are described herein and are known in the art.
[101] The chromatography column (s) and / or chromatographic membrane (s) present in an MCCS can be linked or moved relative to each other by a switching mechanism (for example, a column switching mechanism). The MCCS can also include one or more (for example, two, three, four, or five) pumps (for example, automated, for example, automated peristaltic pumps). Column switching events can be triggered by the detection of a level of protein to be purified detected by UV absorbance corresponding to a certain level of protein in the fluid that passes through the MCCS (for example, the entry and / or eluate of a or more of the chromatography column (s) and / or chromatographic membranes in the MCCS), a specified volume of liquid (eg, buffer), or the specific elapsed time. Column switching generally means a mechanism through which at least two different chromatography columns and / or chromatography membranes in an MCCS (for example, two or more different chromatography columns and / or chromatographic membranes present in an MCCS are allowed to pass through a different step (for example, equilibration, loading, elution, or washing) at substantially the same time at least during part of the process.
[102] The chromatography column (s) and / or the chromatographic membrane (s) present in an MCCS can have one or more of any of the exemplary shapes, sizes, volumes (bed volumes), and / or the operation ( s) of drive described here.
[103] The chromatography column (s) and / or the chromatographic membrane (s) present in an MCCS may contain one or more of any of the exemplary resins described or known in the art. For example, the resin contained in one or more of the chromatography column (s) and / or chromatographic membrane (s) present in the MCCS may be a resin that uses a capture mechanism (for example, protein binding to a capture mechanism , protein binding to the capture mechanism, antibody capture or fragment binding antibody mechanism, substrate binding capture mechanism, cofactor binding capture mechanism, aptamer binding capture mechanism, and / or a capture mechanism for marker binding). The resin contained in one or more of the MCCS chromatography column (s) and / or chromatographic membrane (s) can be a cation exchange resin, an anion exchange resin, a molecular sieve resin, or a hydrophobic interaction resin , or any combination thereof. Other examples of resins that can be used to purify a protein are known in the art, and can be contained in one or more of the chromatography column (s) and / or chromatographic membrane (s) present in the MCCS. The chromatography of column (s) and / or chromatography membranes present in the MCCS may contain the same and / or different resins (for example, any of the resins described herein or known in the art for use in the purification of recombinant proteins).
[104] The chromatography column (s) and / or chromatographic resin (s) present in the MCCS can perform one or more unit operations (for example, capturing a protein, purifying a protein, polishing a protein, inactivating a virus, adjusting the ionic concentration and / or the pH of a liquid containing the protein, or filter out a fluid containing a protein). In the non-limiting examples, the MCCS can perform the unitary operations of capturing a protein from a fluid (for example, a liquid culture medium) and inactivating viruses present in the liquid containing the recombinant therapeutic protein. The MCCS can perform any combination of two of the more unitary operations described herein or known in the art.
[105] An MCCS can be equipped with: one or more (for example, two, three, four, five, six, seven, eight, nine or ten) UV monitors, one or more (for example, two, three, four, five, six, seven, eight, nine or ten) valves, one or more (for example, two, three, four, five, six, seven, eight meters, nine or ten) pH, and / or one or plus (for example, two, three, four, five, six, seven, eight, nine or ten) meters of conductivity. An MCCS can also be equipped with an operating system that uses the software (for example, software based on Unicorn, GE Healthcare, Piscataway, NJ) to detect when a switching column should occur (for example, based on UV absorbance, the volume of liquid, or elapsed time) and affect (trigger) the column switching events. In the examples where MCCS includes one or more UV detectors, the UV detectors can be optionally placed at the entrance of one or more (for example, two, three, four, five, six, seven, eight, nine or ten) of the column from chromatography (s) and / or chromatographic membrane (s) in the MCCS, and / or at the exit of one or more of the column (s) and / or membrane chromatography to MCCS chromatography.
[106] An MCCS may also include one or more (for example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen , twenty, twenty-one, twenty-two, twenty-three, or twenty-four) in-line buffer adjustment reservoir and / or a buffer reservoir (s). In other examples, the MCCS may include one or more (for example, two, three, four, five, or six) rupture tanks that can contain liquid that cannot easily pass to one or more of the chromatography columns and / or chromatographic membranes in the MCCS. The systems described herein may contain one or more break tanks. Other examples of the systems described here do not include a rupture tank.
[107] An MCCS can include an inlet, through which the fluid (for example, a liquid culture medium that is substantially cell-free) can be passed into said MCCS. The entrance can be any structure known in the art for such purposes. It can include, for example, a thread, ribs, or a seal that allows a fluid conduit to be inserted, such that after insertion of the fluid conduit into the inlet, the fluid enters through the MCCS inlet without infiltration. significant amount of fluid out of the inlet. Non-limiting entries that can be used in the present systems are known and would be understood by those skilled in the art. Some examples of the systems provided here also include a bioreactor that is in fluid connection with the MCCS inlet. Any of the exemplary bioreactors described herein or known in the art can be used in the present systems.
[108] The MCCS may include an outlet through which the protein can exit the system. The outlet may include, for example, a thread, ribs, or a seal that allows a fluid conduit to be inserted or a vial designed to contain or store the protein. An outlet may include a surface that can be used to seal a sterile jar or other storage container for the outlet, to allow protein to flow directly into the sterile jar or storage container. Non-limiting commercial establishments that can be used in the present systems are known and would be understood by those skilled in the art.
[109] Some examples of the systems provided here also include a pump system. A pump system can include one or more of the following: one or more (for example, two, three, four, five, six, seven, eight, nine or ten) pumps (for example, any of the pumps described or known herein) in the art), one or more (for example, two, three, four or five) filters (for example, any of the filters described herein or known in the art), one or more (for example, two, three, four, five , six, seven, eight, nine, or ten) UV detectors, and one or more (for example, two, three, four, or five) break tanks.
[110] Some examples of the systems described in this document additionally include another fluid conduit connected to the fluid conduit between the pump and the MCCS inlet, where one end of the additional fluid conduit is fluidly connected to a bioreactor and the the other end is fluidly connected to the fluid conduit between the pump and the inlet. This new fluid conduit may include a filter that is capable of removing cells from the liquid culture medium removed from the bioreactor (for example, the ATF cell retention system). In some instances, this particular fluid conduit may include one or more (for example, two, three, or four) pumps (for example, any of the pumps described herein or known in the art) and / or one or more (for example example, two, three, or four) rupture tanks (for example, any of the exemplary break tanks described here), where this pump (s) and / or break tank (s) are in fluid connection with the fluid present in the fluid conduit.
[111] The systems described here can optionally include a fluid conduit disposed between the final chromatography column or chromatographic membrane and the outlet. The systems described herein may further include one or more filters in fluid connection with the fluid conduit disposed between the end of the chromatography column or chromatographic membrane at and the outlet, so that the filter can remove, for example, precipitated material, particles, or bacteria from the fluid present in the fluid conduit disposed between the final chromatography column or chromatographic membrane in the MCCS and the outlet.
[112] The method of the invention can be performed in continuous mode. In other words, the method of the invention can be a continuous method for the purification of a protein from the solution.
[113] The term "continuous method" or "method in a continuous mode" means a method that continuously feeds fluid through at least part of the system.
[114] By the term "fluid", here is meant any liquid, such as a solution containing the protein to be purified, a buffer or a low pH acidic solution or for viral inactivation.
[115] In a preferred embodiment, the first, second and third matrices are fed continuously through a fluid.
[116] The term "integrated process" means a process that is carried out using structural elements that work cooperatively to achieve a specific result (for example, the generation of a purified protein from a liquid culture medium).
[117] Examples of the integrated process are disclosed in U.S. Provisional Patent Application 61 / 775,060 (hereby incorporated by reference in its entirety).
[118] Scaling up the process of the present invention may also include the use of a switching column and / or increasing the bed volume of each chromatography column.
[119] In addition, the method of the invention can be performed in a closed system, from the first step of the method to the last. In particular, the chromatography steps and, optionally, the filtration step (s) (for example, the nanofiltration step and / or the ultrafiltration and diafiltration step) can be performed in a closed system. In a specific embodiment of the method of the invention, the solution containing the proteins is passed, part by part, along the three chromatography columns, in particular along the three chromatography columns or membrane adsorbers, each passage of a part of the solution corresponding to an execution. The proteins recovered at the end of each assay are then collected and collected. Specific examples of this method are described in Examples 5, 6 and 8. In such a method, the column or membrane adsorber of a chromatography step is used several times, and, optionally, sanitized using for example a sanitation buffer, as defined above, thus allowing to reduce the amount of resin devices or membrane adsorbent, and necessary buffer. For example, a sequence of 3 to 50 passes (for example, 3 to 30 runs, 5 to 25 runs, 10 to 20 runs or 15 runs), can be performed continuously. More specifically, 3, 4, 5, 6, 7 or 8 runs can be performed in continuous mode, followed by sanitation of the 3 columns or membrane adsorbents (for example, using the sanitation buffer). This can be repeated, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.
[120] The process described here can be used to recover the purified proteins. As used herein, "purified" refers to a purity that allows for the effective use of the protein in vitro, ex vivo or in vivo. For a protein to be useful in in vitro, ex vivo, or in vivo applications, it must be substantially free of contaminants, other proteins, and / or chemicals that may interfere with the use of that protein in such applications, or that at least it would be undesirable for inclusion with the protein of interest. Such applications include the preparation of therapeutic compositions, the administration of the protein in a therapeutic composition, and other methods described herein. Preferably, a "purified" protein, as referred to herein, is a protein that can be produced by any method (i.e., by direct purification from a natural source, recombinantly, or synthetically), and that has been purified from of other protein components such that the protein comprises at least about 80% weight / weight of the total protein in a given composition, and more preferably, at least about 85%, and more preferably at least about 90%, and more preferably at least about 91%, and more preferably at least about 92%, and more preferably at least about 93%, and more preferably at least about 94%, and more preferably at least about 95%, and more preferably at least about 96%, and more preferably at least about 97%, and more preferably at least about 98%, and most preferably at least about 99% weight / weight of the total protein in a given composition.
[121] As used herein, "crude protein" refers to a protein that can be produced by any method (that is, by direct purification from a natural source, recombinantly, or synthetically), and that has been purified at from other protein components so that the protein comprises less than about 80% weight / weight of the total protein in a given composition.
[122] In a particular embodiment, the method for purifying a protein from the solution according to the invention comprises: (a) a first chromatography step comprising: (i) passing the equilibration buffer through a column of protein; (ii) passing the solution through the protein A column; (iii) passing the equilibration buffer through the protein A column; (iv) passing the wash buffer through the Protein A column; (v)) passing the equilibration buffer through the protein A column; and (vi) eluting a crude protein eluent from the Protein A column using a first elution buffer; (b) a second chromatography step comprising: (i) passing the equilibration buffer through a multimodal resin chromatography column; (ii) passing the crude protein eluent from step (a) through the multimodal resin chromatography column; (iii) passing the equilibration buffer through the multimodal resin chromatography column; and (iv) eluting a protein eluate from the multimodal resin chromatography column, using a second elution buffer; (c) a third chromatography step comprising: (i) passing the equilibration buffer through an anion exchange chromatography column; (ii) passing the protein eluate from step (b) through the anion exchange chromatography column, in the flow-through mode; and (iii) recovering the purified protein from the passage flow of the anion exchange chromatography column, in which the equilibrium buffer comprises 15 to 25 mM Bis Tris, and 15 to 25 mM NaCl, adjusted to a pH between 7 and 8 with acetic acid, the wash buffer comprises from 15 to 25 mM Bis Tris, and from 0.9 to 1.1 M NaCl adjusted to a pH between 7 and 8 with acetic acid, the first elution buffer comprises 15 to 25 mM Bis Tris, and 15 NaCl 25 mM, adjusted to a pH between 3 and 4 with acetic acid and the second elution buffer comprises 15 to 25 mM Bis Tris, 40 to 50 mM NaCl, and 20 to 30 mM NH4Cl, adjusted to a pH between 7 and 8 with acetic acid.
[123] In another embodiment, the method for purifying a protein from the solution according to the invention comprises: (a) a first chromatography step comprising: (i) passing the equilibration buffer through a column of protein; (ii) passing a part of the solution through the protein A column; (iii) passing the equilibration buffer through the protein A column; (iv) passing the wash buffer through Protein A Column; (v) passing the equilibration buffer through the protein A column; and (vi) eluting a crude protein eluent from the Protein A column using a first elution buffer; (b) a second chromatography step comprising: (i) passing the equilibration buffer through a multimodal resin chromatography column; (ii) passing the crude protein eluent from step (a) through the multimodal resin chromatography column; (iii) passing the equilibration buffer through the multimodal resin chromatography column; and (iv) eluting a protein eluate from the multimodal resin chromatography column, using a second elution buffer; (c) a third chromatography step comprising: (i) passing the equilibration buffer through an anion exchange chromatography column; (ii) passing the protein eluate from step (b) through the anion exchange chromatography column, in the flow-through mode; and (iii) recovering the purified protein from the passage flow of the anion exchange chromatography column, (d) successively renewing steps a), b) and c) with another part of the solution until the entire solution is used, and (e) collecting the purified proteins recovered at the end of each third chromatography step; where the equilibrium buffer comprises 15 to 25 mM Bis Tris, and 15 to 25 mM NaCl, adjusted to a pH between 7 and 8 with acetic acid, the wash buffer comprises 15 to 25 mM Bis Tris , and 0.9 to 1.1 M NaCl adjusted to a pH between 7 and 8 with acetic acid, the first elution buffer comprises 15 to 25 mM Bis Tris, and 15 NaCl 25 mM, adjusted to a pH between 3 and 4 with acetic acid and the second elution buffer comprises 15 to 25 mM Bis Tris, 40 to 50 mM NaCl, and 20 to 30 mM NH4Cl, adjusted to a pH between 7 and 8 with Acetic Acid.
[124] In another particular embodiment, the method for purifying a protein from the solution according to the invention comprises: (a) a first chromatography step comprising: (i) passing the equilibration buffer through an adsorber protein A membrane; (ii) passing the solution through the Protein A membrane adsorber; (iii) passing the equilibration buffer through the Protein A membrane adsorber; (iv) passing the wash buffer through the Protein A membrane adsorber; (v) passing the equilibration buffer through the Protein A membrane adsorber; and (vi) eluting a crude protein eluent from the protein A membrane adsorber, using a first elution buffer; (b) a second chromatography step comprising: (i) passing the equilibration buffer through a cation exchange membrane adsorber; (ii) passing the crude protein eluent from step (a) through the cation exchange membrane adsorber; (iii) passing the equilibration buffer through the cation exchange membrane adsorber; and (iv) eluting a protein eluate from the cation exchange membrane adsorber using a second elution buffer; (c) a third chromatography step comprising: (i) passing the equilibration buffer through an anion exchange membrane adsorber; (ii) passing the protein eluate from step (b) through the anion exchange membrane adsorber in the flow-through mode; and (iii) recovering the purified protein from the anion exchange membrane adsorber throughflow, in which the equilibrium buffer comprises 15 to 25 mM Bis Tris, and 15 to 25 mM NaCl, adjusted to a pH between 7 and 8 with acetic acid, the wash buffer comprises from 15 to 25 mM Bis Tris, and from 0.9 to 1.1 M NaCl adjusted to a pH between 7 and 8 with acetic acid, the first elution buffer comprises 15 to 25 mM Bis Tris, and 15 NaCl 25 mM, adjusted to a pH between 3 and 4 with acetic acid and the second elution buffer comprises 15 to 25 mM Bis Tris, 50 to 150 mM NaCl, and 20 to 30 mM NH4Cl, adjusted to a pH between 6 and 7 with acetic acid.
[125] In another embodiment, the method for purifying a protein from the solution according to the invention comprises: (a) a first chromatography step comprising: (i) passing the equilibration buffer through an adsorber of protein A membrane; (ii) passing a part of the solution through the Protein A membrane adsorber; (iii) passing the equilibration buffer through the Protein A membrane adsorber; (iv) passing the wash buffer through the Protein A membrane adsorber; (v) passing the equilibration buffer through the Protein A membrane adsorber; and (vi) eluting a crude protein eluent from the protein A membrane adsorber, using a first elution buffer; (b) a second chromatography step comprising: (i) passing the equilibration buffer through a cation exchange membrane adsorber; (ii) passing the crude protein eluent from step (a) through the cation exchange membrane adsorber; (iii) passing the equilibration buffer through the cation exchange membrane adsorber; and (iv) eluting a protein eluate from the cation exchange membrane adsorber using a second elution buffer; (c) a third chromatography step comprising: (i) passing the equilibration buffer through an anion exchange membrane adsorber; (ii) passing the protein eluate from step (b) through the anion exchange membrane adsorber in the flow-through mode; and (iii) recovering the purified protein from the anion exchange membrane adsorber bypass flow, (d) successively renewing steps a), b) and c) with another part of the solution until the entire solution is used, and (e) collecting the purified proteins recovered at the end of each third chromatography step; where the equilibrium buffer comprises 15 to 25 mM Bis Tris, and 15 to 25 mM NaCl, adjusted to a pH between 7 and 8 with acetic acid, the wash buffer comprises 15 to 25 mM Bis Tris , and from 0.9 to 1.1 M NaCl adjusted to a pH between 7 and 8 with acetic acid, the first elution buffer comprises 15 to 25 mM Bis Tris, and 15 NaCl 25 mM, adjusted to a pH between 3 and 4 with acetic acid and the second elution buffer comprises 15 to 25 mM Bis Tris, 50 to 150 mM NaCl, and 20 to 30 mM NH4Cl, adjusted to a pH between 6 and 7 with acetic acid.
[126] The method for purifying a protein from the solution may comprise at least one filtration step, such as a nanofiltration step, an ultrafiltration step and / or a diafiltration step. the filtering step (s) can be performed before and / or after the chromatographic steps. When purifying recombinant proteins for pharmaceutical purposes, the chromatographic steps are typically followed by filtration steps. Therefore, the method of the invention may further comprise a nanofiltration step after step (c). An ultrafiltration and diafiltration stage can still be performed after the nanofiltration stage. As used herein, "ultrafiltration" or "UF" refers to a filtration technique using a semipermeable membrane to physically and selectively remove particles and / or ions from a solution based on the particle size and pore size in the membrane UF. As used herein, "nanofiltration" refers to the filtration of a solution through a nanofilter that is used to remove, for example, viral particles. As used herein, "diafiltration" refers to a technique that uses ultrafiltration membranes to completely remove, replace or lower the concentration of salts or solvents from solutions.
[127] The method of the invention may also additionally comprise at least one viral inactivation step. Said at least one viral inactivation step can be carried out at any stage of the method of the invention, for example, before step (a), after step (a), after step (b), after step (c) , after the nanofiltration stage and / or after the ultrafiltration and diafiltration stage. Such a viral inactivation step can typically be a low pH or acidic inactivation step. As used herein, "low pH or acid inactivation" refers to a viral pH inactivation technique with acid pH to denature viruses, in particular encapsulated viruses. Typically, the low or acidic pH inactivation step is carried out by incubating the recovered proteins at a pH of between about 3.0 to 5.0 (for example, between about 3.5 to about 4.5, between about 3.5 to about 4.25, between about 3.5 to about 4.0, for example 4.0) for a period of at least 30 minutes (for example, a period of between 1 hour to 21 days, a period of between about 2 hours to 21 days, or a period of between about 4 hours to 21 days). For example, the acid pH inactivation step is carried out by incubating the recovered proteins at a pH of 4, for example for 6 h to 21 days.
[128] The method of the invention may also comprise, prior to step (a), a step of providing a liquid culture medium containing the protein to be purified, which is substantially cell-free, wherein said liquid culture medium is fed to the first chromatography column.
[129] For example, the method of the invention for purifying a protein from the solution may comprise: (pre-a), a step of providing a liquid culture medium containing the protein to be purified, which is substantially free of cells, (a) a first chromatography step comprising: - passing said liquid culture medium from step (pre-a) through a first chromatography column; - Elute a crude protein eluent from the first chromatography column using a first elution buffer; (b) a second chromatography step comprising: - passing the crude protein eluent obtained at the end of step (a) through a second chromatography column; - Elute a protein eluate from the second chromatography column using a second elution buffer; and (c) a third chromatography step comprising: - Passing the protein eluate obtained at the end of step (b) through a third chromatography column in the flow-through mode; - Recover purified protein from the flow through the third chromatography column; wherein each of the plugs comprises Bis Tris.
[130] Finally, the purified protein can be formulated in a composition suitable for storage, and / or in a pharmaceutical composition suitable for administration to animals and / or humans.
[131] One of the numerous advantages of the disclosed method is that it allows to obtain good yields of highly pure protein. The purified protein that is recovered with the method of the invention can, for example, have a degree of purity of at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5% or 99 , 9%. More particularly, one of the numerous advantages of the disclosed method is that it allows the solutions to obtain highly pure proteins containing quantities of contaminating DNA, high molecular weight species (HMW) (which corresponds to protein aggregates) and / or host cell proteins (HCP). The solution comprising the purified protein that is recovered with the method of the invention may, for example, exhibit an amount of contaminating DNA less than 0.4 ppb, less than 0.3 ppb, less than 0.2 ppb or less than 0, 1 ppb. The solution comprising the purified protein that is recovered with the method of the invention can also, for example, have a concentration of HMW species of less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5% or less than 0.4%. The solution comprising the purified protein that is recovered with the method of the invention may also, for example, have an HCP concentration of less than 23 ppm, less than 22 ppm, less than 21 ppm, less than 20 ppm, less than 19 ppm or less than 18 ppm. In addition, the method of the invention may allow the purified protein to be recovered in a yield of at least 85%, 90%, 95%, 96%, 97%, 98% or 99%.
[132] Another aspect of the invention relates to a method for the preparation of buffers suitable for use in the method of the invention. In fact, all of these buffers can very easily and quickly be prepared from a single stock solution.
[133] Such a method for the preparation of buffers may comprise or consist of the following steps: i) creating a solution (for example, a 100 L solution) with a final concentration of 15 to 25 mM (for example 20 mM) of Bis Tris and 15 to 25 mM (e.g. 20 mM) NaCl; ii) adjust the pH of the solution to a value between 7 and 8 (for example, 7.4) with acetic acid; iii) collect a quarter of the solution, thus obtaining an equilibration buffer; iv) adjusting the pH of a quarter of the remaining three quarters of solution from step (iii) to a value between 3 and 4 (for example 3.7) with acetic acid, thus obtaining an elution buffer; v) collect a quarter of the remaining two quarters of the solution from step (iii), add NaCl to obtain a final concentration of NaCl between 40 and 50 mM (for example 45 mM), add more NH4Cl to obtain a final NH4Cl concentration between 20 and 30 mM (for example 25 mM), and adjust the pH to a value between 7 and 8 (for example, 7.25) with acetic acid, thus obtaining an additional elution buffer; vi) add NaCl to the remainder of the quarter of the solution from step (iii), and add NaCl to obtain a final concentration of NaCl between 0.9 to 1.1 M (for example, 1 M), obtaining thus a wash buffer.
[134] Such a method is represented schematically in Figure 1.
[135] Another method for the preparation of buffers may comprise or consist of the following steps: i) creating a solution (for example, a 100 L solution) with a final concentration of 15 to 25 mM (for example 20 mM) of Bis Tris and 15 to 25 mM (e.g. 20 mM) NaCl; ii) adjust the pH of the solution to a value between 7 and 8 (for example, 7.4) with acetic acid; iii) collect a quarter of the solution, thus obtaining an equilibration buffer; iv) adjusting the pH of one quarter of the remaining three quarters of solution from step (iii) to a value between 3 and 4 (for example 3.7) with acetic acid, thus obtaining an elution buffer; v) collect a quarter of the remaining two quarters of the solution from step (iii), add NaCl to obtain a final concentration of NaCl between 70 and 90 mM (for example 80 mM), add more NH4Cl to obtain a final concentration of NH4Cl between 20 and 30 mM (for example 25 mM), and adjust the pH to a value between 6 and 7 (for example 6.2) with acetic acid, thus obtaining an additional elution buffer; vi) add NaCl to the remainder of the quarter of the solution from step (iii), and add NaCl to obtain a final concentration of NaCl between 0.9 to 1.1 M (for example, 1 M), obtaining thus a wash buffer.
[136] The above method for the preparation of buffers can also correspond to the first step of the method of the invention, before carrying out the three chromatographic steps.
[137] The invention further relates to a kit comprising or consisting of: (a) a multimodal resin or cation exchange chromatography column or, an affinity chromatography column, such as a Protein A matrix, and / or an anion exchange chromatography column; and (b) at least one buffer according to the invention (for example, comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl), and / or instructions for the preparation of at least one buffer according to the invention (for example, comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl).
[138] In one embodiment, the kit comprises or consists of: (a) a multimodal resin chromatography column, an affinity chromatography column, such as a protein column, and / or an anion exchange chromatography column; and (b) at least one buffer according to the invention (for example, comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl), and / or instructions for the preparation of at least one buffer according to the invention (for example, comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl).
[139] In another embodiment, the kit comprises or consists of: (a) a cation exchange membrane adsorber, affinity chromatography membrane adsorber such as a Protein A membrane adsorbor, and / or a membrane adsorber anion exchange chromatography; and (b) at least one buffer according to the invention (for example, comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl), and / or instructions for the preparation of at least one buffer according to the invention (for example, comprising or consisting of Bis Tris, acetic acid, NaCl, water, and, optionally, NH4Cl). EXAMPLES
[140] The following Examples are illustrative of specific modalities of the disclosed method, and various uses of these. They are presented for explanatory purposes only and are not to be construed as limiting the scope of the invention in any way. Example 1: Optimization of Purification Buffers
[141] Bis Tris buffers can advantageously be used as buffers with a multimodal resin chromatography column.
[142] The crude protein eluent obtained after passing through a Protein A chromatography column was passed through a Capto MMC multimodal resin chromatography column.
[143] This resin is capable of correcting mAb even at low pH, such as those obtained after a chromatography step on a protein column. However, this resin also needs salt to obtain a good elution. However, too high a concentration of salt is detrimental to mAb stability, since it increases the level of high molecular weight (HMW) species in eluted samples, and is very efficient in fixed impurities in the medium, leading to a decrease in the purity of the mAb.
[144] To avoid these disadvantages, the inventors carried out an experiment design (DOE) in order to reduce the concentration of salt and pH in the elution buffer of step (a).
[145] To this end, the inventors introduced a new type of salt, ammonium chloride, which has the same conductivity as NaCl, but is stronger in terms of Na + interactions. DOE parameters
[146] A central composite side (CCF) design was applied to optimize the elution conditions with MODDE 9 software (UMETRICS). The CCF project was composed of a complete factorial design and three central points (in all 17 experiments). The pH elution varied between 7.2 and 7.8, and in the same way the concentration of NaCl was varied from 0 to 100 mM and the concentration of NH4CI was varied from 0 to 50 mM.
Abbreviations: Quant .: quantitative; Control .: controlled; Transfer: Transformation; Prec: precision; MLR: multiple linear regression; Orthog .: Orthogonal; PLS: partial least squares; Var. Unit: unit variance; HCP: host cell protein ;: HMW: high molecular weight species DOE experiments
[147] The 17 experiments are summarized in the table below.
DOE Results
[148] It appeared during the runs that four experiments were inconsistent in terms of product recovery. N1 and N2 runs had no elution due to conditions and during N5 and N11 runs, elution had to be stopped after 10 column volumes (CV). These experiments were excluded from the analysis.
[149] All experiments were subjected to SEC-HPLC and HCP analysis.
[150] The results are summarized in the table below.

[151] In the MODDE 9.0 forecasting tool, the inventors determined the sweet spot, that is, the space containing the conditions to apply, so the following responses are fixed: - Yield: 95 to 100% - HCP: 20 to 30 ppm - HMW: 0.4 to 0.8%
[152] The sweet spots are shown in Figure 2, for three types of NH4Cl concentration (0, 25 and 50 mM).
[153] The black space is the space that contains the first criterion met: a yield between 90 and 100%. The light grayish space is the space that contains both the yield and the HCP rate (20 to 30 ppm). The dark gray space is the sweet spot that is the space that contains the 3 criteria met: productivity, HCP rate and HMW rate (0.4 to 0.8%).
[154] The inventors thus demonstrated, with this graphical description, that the more NaCl was used, the more HMW and yield were increased. In addition, the higher the pH, the more the HCP rate was important. Therefore, the inventors demonstrated that, in order to obtain a high yield, with a good purity, the elution had to be carried out at a low pH and with a low concentration of NaCl.
[155] The inventors have demonstrated that NH4Cl can be used to decrease the concentration of NaCl. The most suitable NH4CI concentration was 25 mM, giving a sweet spot with more than 95% yield, a rate of about 30 ppm HCP and a rate of 0.6% HMW.
[156] The inventors thus demonstrated that the ideal elution buffer for the second chromatography step was 20 mM Bis Tris, 45 mM NaCl, 25 mM NH4Cl, pH acetic acid qsp 7.25. Example 2: Optimization of the third chromatographic step
[157] A purification process, in which the Sartobind STIC membrane was used as a third step, due to its tolerant property, in order to polish impurities after the first two chromatographic steps and to remove viruses, was initially designed.
[158] However, in a continuous process, each step is performed several times. Disposable membranes, such as Sartobind STIC membranes, cannot be reused. The inventors, therefore, designed a third stage of alternative chromatography allowing polishing of impurities, removal of viruses and being reusable, which can thus be used in a continuous process.
[159] Three types of media were tested as a polishing step: - Sartobind STIC (Sartorius) - Sartobind Q (Sartorius), and - BioPro Q75 (YMC), an AEX resin.
[160] First, the inventors tested these resins after an MMC Capto elution (100 mM NaCl, 20 mM Bis Tris, pH 8.0) to assess the ability of the third step to remove impurities, even under conditions of 100 mM NaCl. The Sartobind STIC and Q membranes were evaluated in a single run. BioPro Q75 was evaluated in three experiments: concentration of 100 mM, 50 mM and 25 mM NaCl. The results are shown in the table below.

[161] These results show, therefore, that the Sartobind STIC membrane, due to its salt-tolerant property, gives good removal of impurities. However, an increase in pressure during operation from 1 to 3 bars, shows that it is not possible to use such membranes in a reusable technology, again, the pressure has not decreased after a 1 M NaCl wash and after cleaning NaCl at 0.1 N. The best results in terms of impurities were obtained with the Sartobind Q membrane. However, due to issues of reuse and availability, this membrane was considered as not suitable by the inventors.
[162] Finally, BioPro Q75 resin gave intermediate results in terms of removing impurities. In addition, no pressure was observed during the executions. Finally, reusability has been completed due to classic resin technology.
[163] The inventors thus demonstrated that an anion exchange chromatography column was a good alternative to the membrane in polishing steps, in order to carry out the purification of the protein by a continuous process. Example 3: Formulation of Purification Buffers
[164] The three-step purification method described here uses four buffers: one equilibration buffer, wash buffer, and two elution buffers, all prepared from the same mother solution. An outline of the protocol is shown in Figure 1 and is as follows: eq. 20 mM Tris and Bis eq. 20 mM NaCl was brought to 100L of water for injection (WFI) as the mother solution, and the pH of the solution was then adjusted to 7.4 using acetic acid. 25 L of the resulting solution was then collected and stored as the equilibration buffer. 25L of the stock solution was then removed and eq. 1 M NaCl was added. This resulting 25L solution was the wash buffer. The pH of 25L of the stock solution was then adjusted to 3.7 with acetic acid. This resulting 25L solution was the first elution buffer. The remaining 25 L of the stock solution was then pH adjusted to 7.25 using acetic acid, and eq. 45mM of NaCl eq. 25 mM NH4Cl was added, resulting in the other elution buffer. Example 4: Continuous Small-Scale Multi-step Process
[165] The method of the invention was used for purification in small batches of a humanized monoclonal antibody that specifically binds to the transmembrane glycoprotein CD38 (anti-CD38 mAb). Materials and methods Material - First stage: 60 mL of Absolute High Cap resin in an XK50 / 20 column customized with Fast Flow Tubing - Second stage: 100 mL of Capto MMC resin in an XK50 / 20 column personalized with Fast Flow tubing - Third stage : 50 mL of BioPro Q75 resin in a customized XK50 / 20 column with Fast Flow tubing - Modified Akta Purifier tubing with PEEK id adjustment 1.0 mm Details of the first stage The XK50 / 20 column was packed with Absolute High Cap resin (Ref. AbSHC 35 P1-AV-00200). The final volume was 60 mL of HETP was 13538 N / m and asymmetry was 1.2. Three types of buffer were used for this step: - Balance buffer made with 20 mM Bis Tris, 2 mM NaCl, pH 7.4 in qsp acetic acid - Wash buffer made with 20 mM Bis Tris, 1 M NaCI, acetic acid pH 7.4 qsp - Elution buffer made with 20 mM Tris Bis, 20 mM NaCl, qsp acetic acid pH 3.7. The flow rate, according to the column size, was created at 24 mL / min (RT 2.5 min) for the equilibration, washing, loading and elution step. Second stage details The XK50 / 20 column was packed with MMC Capto resin (Ref 17-5317-02). The final volume was 100 ml. From HETP it was 5686 N / m and asymmetry was 1.3. Two types of buffer were used for this step: - Balance buffer made with 40 mM Bis Tris, 20 mM NaCl, pH 7.4 acetic acid qsp - Elution buffer made with 20 mM Tris Bis, 45 mm NaCl, 25 mM NH4Cl, pH acetic acid qsp 7.25. The flow rate, according to the column size, was adjusted to 24 mL / min (RT 4.8 min) to achieve equilibrium, load and elution step. Details of the Third Stage The XK50 / 20 column was packed with BioPro Q75 resin (Ref QAA0S75). The final volume was 50 mL of HETP was 4875 N / m and asymmetry was 1.6. A type of buffer was used for this step: - Balance buffer made with 40 mM Bis Tris, 20 mM NaCl, acetic acid qsp pH 7.4. The flow rate, according to the column size, was adjusted to 24 mL / min (RT 2.1 min) for the equilibration and elution step. Results of
[166] 2,325 g of mass collection (at a concentration of 1.71 g / L) were loaded on Absolute High Cap resin. The total duration to purify the 2325 g was 2h10min. 2.125 g were recovered which means that the yield was 91%. Technically, purification was successfully achieved without back pressure, even though the columns were used in a series configuration.
[167] The removal of impurities from the final product obtained with this 3-step process is summarized and compared with that obtained with the 2-step process described in document PCT / EP2012 / 059528, in the table below.

[168] The inventors thus showed that the new continuous multi-step process was as efficient as the 2-step process described in document PCT / EP2012 / 059528, applied through the same product.
[169] Small-scale studies were carried out to evaluate the rate of impurities obtained after each stage. The inventors showed that each step was efficient for removing impurities, as summarized in the following table.

[170] This example demonstrates, therefore, that the continuous process devised by the inventors is the most efficient as a batch process. Example 5: Continuous Full Scale Multi-step Process
[171] The above method has been applied to large-scale purification of a humanized monoclonal antibody that specifically binds to the CD38 transmembrane glycoprotein (anti-CD38 mAb). Materials and methods Materials - First stage: 50 mL of Absolute High Cap resin with a dynamic binding capacity (DBC) of 50 mg / mL - Second stage: 100 mL of MMC Capto resin with a DBC of 35 mg / mL - Third stage : 50 mL of BioPro Q75 resin with a 170 mg / mL DBC - GE Periodic Countercurrent system (customized Akta purifier), comprising the 3 columns above in a row and allowing the monitoring of all columns. Methods A sequence of 15 executions was performed continuously. More specifically, 5 experiments were carried out in continuous mode followed by sanitation of columns 3, with 0.1 N NaOH, before starting a new sequence. Results of
[172] One sequence made it possible to purify 10g of anti-CD38 mAb in less than 500 min.
[173] 29.5 g of bulk collection was loaded and 28 g of purified mAb was recovered. The continuous multistep process of the invention, therefore, allowed an average yield of 95% to be achieved. These 28 g of purified mAb were obtained in 25h.
[174] In addition, the results of the analysis of the final product were comparable to those of the final product obtained with the 2-step process described in document PCT / EP2012 / 059528. These analytical results are summarized in the table below.

[175] This was also the case when each step was analyzed separately, as shown in the table below.


[176] In addition, as shown in Figure 3, the trends do not show any significant differences between each run. Example 6: Batch Purification in Continuous Mode
[177] The process described in Example 5 was used to purify the anti-CD38 mAb in a full-scale continuous batch.
[178] In this example, 43L of mAb, at a concentration of 1.66 g / L, was purified continuously for 69 h. More specifically, 45 runs were performed, 960 ml of clarified product being loaded in each run. This led to the recovery of 19.5 L of purified mAb, at a concentration of 3.42 g / L, which represents a yield of 93%, using only 70 L of buffers.
[179] The table below summarizes the characteristics of the present purification method compared to those of a 2-step purification process described in document PCT / EP2012 / 059528.

[180] Consequently, the continuous multistep process of the invention allows the reduction of the volume of used buffers by 33% and the volume of resins used by 97%. Example 7: Purification of Different Monoclonal Antibodies
[181] In addition to the humanized anti-CD38 antibody, the continuous multi-step process described above was used to purify additional antibodies, that is, a fully human antibody that specifically binds to the bacterial surface polysaccharide poly-N-acetyl glucosamine (PNAG ), a monoclonal antibody that specifically binds to the cell adhesion molecule related to carcinoembryonic antigen 5 (CEACAM5) and humanized mAb 13C3, which binds to the protofibrillary form of human β-amyloid protein, as described in International Publication No. WO 2009/065054
[182] The table below shows the overall yield and the purity obtained by purifying these three antibodies.
1 The general yield corresponds to the yield before the nanofiltration, ultrafiltration and diafiltration steps.
[183] In conclusion, it has been confirmed with four different antibodies that the continuous multi-step method method allows to obtain good yields of purified antibodies with an excellent degree of purity, purified antibodies with a quality suitable for administration to humans. Example 8: Continuous Multi-step Process of Membrane Adsorbers
[184] The method of the invention using disposable membrane adsorbers has been applied to large-scale purification of a humanized monoclonal antibody that specifically binds to CD38 (anti-CD38 mAb). Materials and methods Materials - First step: 4 Protein A membrane adsorbers Sartobind (Sartorius) of 2 ml each - Second step: 2 membrane adsorbents Sartobind S "nano" (Sartorius) of 3 ml each - Third step: 1 adsorber of 3 mL Sartobind Q "nano" (Sartorisu) membrane The buffers used were the same as those described in Example 4, with the exception of the elution buffer from the second step made with 20 mM Bis Tris, 80 mM NaCl, 25 mM NH4Cl, qsp pH acetic acid 6.2. Methods
[185] The process was carried out to purify 1.5 g of antibodies through 50 runs of 30 mg each through 3 steps above in a continuous mode.
[186] Briefly, the Protein A membrane adsorber was equilibrated with the equilibration buffer, then loaded. After loading, the membrane adsorber was equilibrated again, before elution with the first elution buffer. The protein A membrane adsorbent eluate was directly loaded through the cation exchange membrane adsorbent. While the cation exchange membrane was equilibrated with the equilibration buffer, the Protein A membrane adsorber was sanitized with the sanitation buffer before a next charge. The cation exchange membrane adsorber was eluted with the second elution buffer and the eluate was loaded directly into the anion exchange membrane adsorber. Results of
[187] It was possible to purify 1.5 g of antibodies in 750 min (15 min run). The recovery was about 80%.
[188] The analytical results are summarized in the table below.

[189] Therefore, the continuous multistep process according to the invention, using disposable membrane adsorbers, allows satisfactory impurity removal rates to be obtained, within the internal specifications, without any other optimization of the process and buffers in comparison with the process of using reusable resins.
[190] Furthermore, as shown in Figure 4, the trends did not show any significant differences between each trial. Therefore, the inventors surprisingly demonstrated that disposable membrane adsorbents were in fact stable and can be reused through 50 runs without any decrease in performance.
[191] The main advantages of using membrane adsorbents, instead of columns in the method of the invention, are summarized below: - on a comparable scale, membrane adsorbents can be used at a flow rate 10 times greater than a column , thereby dramatically reducing the duration of the process. For example, a 5 ml column, packed with resin will be used at a flow rate of 1 ml / min, while a corresponding 5 ml membrane adsorber will be used at a minimum flow rate of 10 ml / min. Consequently, when the 3-stage chromatographic process of the invention is carried out in 2h30 with 3 columns filled with resins, it can be completed in 15 min, using membrane adsorbents. - even if they are reusable, membrane adsorbents are disposable devices, which can thus be discarded after a batch and do not need to be stored for a long time. Therefore, it is not necessary to test them to ensure long-term stability. - the method of the invention using membrane adsorbents is cheaper, avoiding column cost, column packing and column storage. Example 9: Continuous multi-stage process of GMP scale
[192] The above method has been applied to large-scale purification of a humanized monoclonal antibody that specifically binds to the CD38 transmembrane glycoprotein (anti-CD38 mAb). Materials and methods Materials - First stage: 6L MabSelect Sure resin with a 35 mg / mL dynamic bonding capacity (DBC) - Second stage: Capto MMC 6L resin with a 35 mg / mL DBC - Third stage: BioPro Q75 6L resin with a 170 mg / mL DBC - 2 AktaProcess from GE (pilot the 3 columns) and a Flexact from Sartorius (completing a low pH inactivation stage between the first stage and the second stage), comprising the 3 columns above in line and that allows the control of all the columns. Methods
[193] The sequence of 7 executions was carried out continuously. Results of
[194] One sequence allowed purification of 1.3 kg of anti-CD38 mAb in less than 16 h.
[195] 14.4 kg of bulk collection was loaded and 1.30 kg of purified mAb was recovered. The continuous multistep process of the invention, therefore, allowed an average yield of 90% to be achieved. This 1.3 kg of purified mAb was obtained in 16 h. For the sake of comparison, the purification of a batch of 500 liters with a two-stage process takes 3 days and uses 20L columns. The continuous approach according to the invention is, therefore, an evolution that saves time and raw materials (saves more than 66% of the volume of resins).
[196] In addition, the results of the analysis of the final product were comparable to those of the final product obtained with the 2-step process described in document PCT / EP2012 / 059528. These analytical results are summarized in the table below.

权利要求:
Claims (17)
[0001]
1. Method for the purification of a protein from a solution, characterized by the fact that it comprises: (a) a first chromatography step comprising: - passing said solution through a first chromatography column, said first column of chromatography being an affinity chromatography column which is a Protein A column; - eluting a crude protein eluent from the first chromatography column using a first elution buffer; (b) a second chromatography step comprising: - passing the crude protein eluent obtained at the end of step (a) through a second chromatography column, said second chromatography column being a multimodal resin chromatography column; - eluting a protein eluate from the second chromatography column using a second elution buffer; and (c) a third chromatography step comprising: - passing the protein eluate obtained at the end of step (b) through a third chromatography column in flow-through mode, said third chromatography column being a chromatography column anion exchange; - recovering purified protein from the passage flow of the third chromatography column; in which each of the buffers consists of Bis Tris, acetic acid, NaCl, water and optionally NH4Cl, and in which said method is carried out continuously.
[0002]
2. Method according to claim 1, characterized by the fact that the crude protein eluent obtained at the end of step (a) is passed directly through a second chromatography column, without undergoing any treatment, such as adjustment of pH, buffer change or dilution.
[0003]
3. Method according to claim 1 or 2, characterized by the fact that the protein eluate obtained at the end of step (b) is passed directly through a third column of chromatography, without undergoing any treatment, such as adjustment pH, buffer change or dilution.
[0004]
Method according to any one of claims 1 to 3, characterized in that the method is carried out in a closed system from the first step to the last.
[0005]
Method according to any one of claims 1 to 4, characterized in that each of the first two chromatography steps comprises: - passing equilibration buffer through the chromatography column; - passing the crude protein solution or eluent through the chromatography column; - pass equilibration buffer through the chromatography column; - optionally pass wash buffer through the chromatography column; - optionally pass equilibration buffer through the chromatography column; - eluting the crude protein eluent or the protein eluate from the chromatography column using an elution buffer, each buffer consisting of Bis Tris, acetic acid, NaCl, water and optionally NH4Cl.
[0006]
Method according to any one of claims 1 to 5, characterized in that said method comprises the steps of: (a) a first chromatography step comprising: (i) passing equilibration buffer through a first column of chromatography, said first chromatography column being an affinity chromatography column which is a Protein A column; (ii) passing the solution through the first chromatography column; (iii) passing equilibration buffer through the first chromatography column; (iv) passing wash buffer through the first chromatography column; (v) passing equilibration buffer through the first chromatography column; and (vi) eluting a crude protein eluent from the first chromatography column using a first elution buffer; (b) a second chromatography step comprising: (i) passing equilibration buffer through a second chromatography column, said second chromatography column being a multimodal resin chromatography column; (ii) passing the crude protein eluent from step (a), through the second chromatography column; (iii) equilibrium passage buffer through the second chromatography column; and (iv) eluting a protein eluate from the second chromatography column using a second elution buffer; and (c) a third chromatography step comprising: (i) passing equilibration buffer through a third chromatography column, said third chromatography column being an anion exchange chromatography column; (ii) passing the protein eluate from step (b) through the third chromatography column in flow-through mode; (iii) optionally passing wash buffer through the third chromatography column; and (iv) recovering purified protein from the passage flow of the third chromatography column.
[0007]
Method according to any one of claims 1 to 6, characterized in that the protein is a monoclonal antibody.
[0008]
8. Method according to claim 7, characterized in that said monoclonal antibody is selected from the group consisting of an antibody that specifically binds to the protofibrillar form of human β-amyloid protein, an antibody that specifically binds to poly -N-acetyl glucosamine of bacterial surface polysaccharide (PNAG), an antibody that specifically binds to the cell adhesion molecule related to the carcinoembryonic antigen 5 (CEACAM5) and an antibody that specifically binds to the transmembrane glycoprotein CD38.
[0009]
Method according to any one of claims 1 to 8, characterized in that it additionally comprises a nanofiltration step after step (c).
[0010]
10. Method according to claim 9, characterized in that it additionally comprises an ultrafiltration and diafiltration step after the nanofiltration step.
[0011]
Method according to any one of claims 1 to 10, characterized in that the first elution buffer comprises 15 to 25 mM Bis Tris, and 15 to 25 mM NaCl, adjusted to a pH between 3 and 4 with acetic acid.
[0012]
Method according to any one of claims 1 to 11, characterized in that the second elution buffer comprises 15 to 25 mM Bis Tris, 40 to 50 mM NaCl, and 20 to 30 mM NH4Cl, adjusted at a pH between 7 and 8 with acetic acid.
[0013]
Method according to any one of claims 5 to 12, characterized in that the equilibrium buffer comprises 15 to 25 mM Bis Tris, and 15 to 25 mM NaCl, adjusted to a pH between 7 and 8 with acetic acid.
[0014]
Method according to any one of claims 5 to 13, characterized in that the wash buffer comprises 15 to 25 mM Bis Tris, and 0.9 to 1.1 M NaCl adjusted to a pH between 7 and 8 with acetic acid.
[0015]
Method according to any one of claims 1 to 14, characterized in that the purified protein is recovered in a yield of at least 95%.
[0016]
16. Method according to any one of claims 1 to 15, characterized in that the recovered purified protein exhibits a purity of at least 99%.
[0017]
17. Method according to any one of claims 1 to 16, characterized in that it additionally comprises the step of formulating the purified protein recovered in a pharmaceutical composition.

类似技术:

---

公开号 | 公开日 | 专利标题

---

BR112015027812B1|2021-03-30|METHOD FOR PURIFYING A PROTEIN FROM A SOLUTION

---

AU2017245478B2|2019-05-16|Protein purification using Bis-Tris buffer

---

US20210009634A1|2021-01-14|Full flow-through process for purifying recombinant proteins

---

EP2782925A1|2014-10-01|Protein purification using bis-tris buffer

同族专利:

---

公开号 | 公开日

---

TW201908331A|2019-03-01|

---

EP2994485A1|2016-03-16|

---

TWI631132B|2018-08-01|

---

US10793622B2|2020-10-06|

---

MX2015015482A|2016-03-21|

---

AU2014264697B2|2019-08-22|

---

EP3301115B1|2021-11-10|

---

KR20160005047A|2016-01-13|

---

RU2015151869A|2017-06-13|

---

MX368177B|2019-09-23|

---

JP2019104735A|2019-06-27|

---

CA2911462A1|2014-11-13|

---

TW201522363A|2015-06-16|

---

WO2014180852A1|2014-11-13|

---

TWI675041B|2019-10-21|

---

KR102272851B1|2021-07-02|

---

JP2016519137A|2016-06-30|

---

DK2994485T3|2018-04-23|

---

JP6463734B2|2019-02-06|

---

RU2662668C2|2018-07-26|

---

IL242408A|2020-04-30|

---

EP3301115A1|2018-04-04|

---

CN105358572A|2016-02-24|

---

NO2994485T3|2018-06-09|

---

HUE038741T2|2018-11-28|

---

CA2911462C|2021-04-06|

---

SG11201508968XA|2015-11-27|

---

AU2014264697A1|2015-11-26|

---

DK3301115T3|2022-01-31|

---

ES2665645T3|2018-04-26|

---

US20160083454A1|2016-03-24|

---

BR112015027812A2|2017-07-25|

---

EP2994485B1|2018-01-10|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

KR930008447B1|1987-10-23|1993-09-04|쉐링 코포레이션|Method of purifying protein|

---

CA2106612C|1993-09-21|2001-02-06|Diana Pliura|Displacement chromatography process|

---

ES2126775T3|1993-09-21|1999-04-01|Hemosol Inc|CHROMATOGRAPHIC PROCEDURE OF DISPLACEMENT AND PRODUCT OF PURIFIED HEMOGLOBIN.|

---

US5891741A|1997-05-16|1999-04-06|Coulter International Corp.|Antibody-aminodextran-phycobiliprotein conjugates|

---

CN1136317C|1999-03-12|2004-01-28|中国人民解放军第四军医大学|Process for preparing bisegmentum and monosegmentum of monoclone antibody|

---

US20020009445A1|2000-07-12|2002-01-24|Yansheng Du|Human beta-amyloid antibody and use thereof for treatment of alzheimer's disease|

---

US6602855B2|2001-04-30|2003-08-05|Syn X Pharma, Inc.|Biopolymer marker indicative of disease state having a molecular weight of 1449 daltons|

---

CN1504482A|2002-12-03|2004-06-16|北京优力凯生物技术有限责任公司|Separation and purification process for acellular whooping cough antigen albumen|

---

PT1745075E|2004-04-21|2013-06-17|Brigham & Womens Hospital|Poly-n-acetyl glucosamine -binding peptides and methods of use thereof|

---

AU2005296352B2|2004-10-21|2011-01-06|Cytiva Bioprocess R&D Ab|Chromatography ligand|

---

NZ561883A|2005-03-23|2010-11-26|Genmab As|Antibodies against CD38 for treatment of multiple myeloma|

---

CN1814775A|2005-12-06|2006-08-09|中国海洋大学|Method for preparing antiviral active oligomeric peptide|

---

EP1914242A1|2006-10-19|2008-04-23|Sanofi-Aventis|Novel anti-CD38 antibodies for the treatment of cancer|

---

RU2493163C2|2007-07-11|2013-09-20|Ново Нордиск А/С|Method of purifying coagulation factor viii protein and method of stabilising factor viii protein|

---

MX2010003828A|2007-11-16|2010-04-27|Univ Rockefeller|Antibodies specific for the protofibril form of beta-amyloid protein.|

---

TWI472339B|2008-01-30|2015-02-11|Genentech Inc|Composition comprising antibody that binds to domain ii of her2 and acidic variants thereof|

---

EA201070987A1|2008-02-29|2011-04-29|Байоджен Айдек Ма Инк.|PURIFIED HYBRID PROTEINS OF IMMUNOGLOBULIN AND METHODS OF THEIR PURIFICATION|

---

EP2281000A2|2008-05-15|2011-02-09|Novo Nordisk A/S|Antibody purification process|

---

MX2011000666A|2008-07-18|2011-03-21|Talecris Biotherapeutics Inc|Method of preparing alpha-1 proteinase inhibitor.|

---

US8497358B2|2008-12-19|2013-07-30|Takeda Pharmaceutical Company Limited|Antibody purification method|

---

AU2010205007A1|2009-01-13|2011-07-07|Ge Healthcare Bio-Sciences Ab|Precipitation of biomolecules with negatively charged polymers|

---

KR101224468B1|2009-05-20|2013-01-23|주식회사 파멥신|Bispecific antibody having a novel form and use thereof|

---

ES2817802T3|2009-08-07|2021-04-08|Emd Millipore Corp|Methods to purify a target protein from one or more impurities in a sample|

---

US9527010B2|2009-09-25|2016-12-27|Ge Healthcare Bio-Sciences Corp.|Separation system and method|

---

US20120208986A1|2009-10-20|2012-08-16|Wenger Marc D|Use of mixed mode chromatography for the capture and purification of basic antibody products|

---

US20130178608A1|2009-12-29|2013-07-11|Samir Kulkarni|Protein purification by ion exchange|

---

EP2415779B1|2010-08-02|2015-01-21|ratiopharm GmbH|Method of producing and purifying an active soluble sialyltransferase|

---

WO2012125735A1|2011-03-15|2012-09-20|Abott Laboratories|An integrated approach to the isolation and purification of antibodies|

---

CN103547589A|2011-03-29|2014-01-29|葛兰素史密斯克莱有限责任公司|Buffer system for protein purification|

---

CA2839542A1|2011-06-17|2012-12-20|Constitution Medical, Inc.|Fixative and staining solutions|

---

WO2013028330A2|2011-08-19|2013-02-28|Emd Millipore Corporation|Methods of reducing level of one of more impurities in a sample during protein purification|

---

EP2578286A1|2011-10-04|2013-04-10|Merck Patent GmbH|Method and apparatus for chromatographic purification|

---

CN107233569B|2011-10-25|2021-08-31|普罗西纳生物科学有限公司|Antibody formulations and methods|

---

WO2013075740A1|2011-11-23|2013-05-30|Sanofi|Antibody purification method|US9950282B2|2012-03-15|2018-04-24|Flodesign Sonics, Inc.|Electronic configuration and control for acoustic standing wave generation|

---

US10967298B2|2012-03-15|2021-04-06|Flodesign Sonics, Inc.|Driver and control for variable impedence load|

---

US10704021B2|2012-03-15|2020-07-07|Flodesign Sonics, Inc.|Acoustic perfusion devices|

---

US9458450B2|2012-03-15|2016-10-04|Flodesign Sonics, Inc.|Acoustophoretic separation technology using multi-dimensional standing waves|

---

JPWO2015041218A1|2013-09-17|2017-03-02|株式会社カネカ|Novel antibody purification method and antibody obtained therefrom , and novel antibody purification method using cation exchange group and novel antibody purification antibody method|

---

US9725710B2|2014-01-08|2017-08-08|Flodesign Sonics, Inc.|Acoustophoresis device with dual acoustophoretic chamber|

---

GB201503578D0|2015-03-03|2015-04-15|Ge Healthcare Bio Sciences Ab|Sanitization method for affinity chromatography matrices|

---

US11021699B2|2015-04-29|2021-06-01|FioDesign Sonics, Inc.|Separation using angled acoustic waves|

---

EP3015542A1|2015-05-07|2016-05-04|Bayer Technology Services GmbH|Modular system and method for continuous, germ reduced production and/or processing of a product|

---

US20170101437A1|2015-10-09|2017-04-13|Hetero Drugs Ltd.|Process for purification of darbepoetin alfa|

---

US11085035B2|2016-05-03|2021-08-10|Flodesign Sonics, Inc.|Therapeutic cell washing, concentration, and separation utilizing acoustophoresis|

---

US11214789B2|2016-05-03|2022-01-04|Flodesign Sonics, Inc.|Concentration and washing of particles with acoustics|

---

WO2018051348A1|2016-09-14|2018-03-22|Insight Biopharmaceutical Ltd.|Methods of purifying and qualifying antibodies|

---

KR102286260B1|2017-08-31|2021-08-05|주식회사 녹십자|How to purify a sulfatase protein|

---

KR20200089334A|2017-12-14|2020-07-24|프로디자인 소닉스, 인크.|Acoustic transducer driver and controller|

---

CN111902720A|2018-03-21|2020-11-06|沃特世科技公司|Non-antibody high affinity based sample preparation, adsorbents, devices and methods|

---

EP3636657A1|2018-10-08|2020-04-15|Ablynx N.V.|Chromatography-free antibody purification method|

---

WO2020084503A1|2018-10-26|2020-04-30|Cadila Healthcare Limited|A composition comprising antibody with reduced level of basic variants thereof|

---

WO2020183332A1|2019-03-11|2020-09-17|Intas Pharmaceuticals Ltd.|Purification of adalimumab using tandem chromatography|

---

EP3947705A1|2019-04-03|2022-02-09|Genzyme Corporation|Continuous production of recombinant proteins|

---

WO2020258704A1|2019-06-28|2020-12-30|信达生物制药有限公司|Seamless continuous flow chromatographic method|

法律状态:
2018-02-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2019-07-09| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NAO 10196/2001, QUE MODIFICOU A LEI NAO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUAANCIA PRA VIA DA ANVISA. CONSIDERANDO A APROVAA AO DOS TERMOS DO PARECER NAO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NAO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDAANCIAS CABA-VEIS. |

---

2020-02-11| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

---

2020-05-26| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2021-02-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2021-03-30| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/05/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

EP13305593.9|2013-05-06|

---

EP13305593|2013-05-06|

---

PCT/EP2014/059246|WO2014180852A1|2013-05-06|2014-05-06|Continuous multistep process for purifying antibodies|

---