WO2021197051A1 - 高效分离纯化重组人凝血因子VIII Fc融合蛋白的方法 - Google Patents

高效分离纯化重组人凝血因子VIII Fc融合蛋白的方法 Download PDF

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WO2021197051A1
WO2021197051A1 PCT/CN2021/081056 CN2021081056W WO2021197051A1 WO 2021197051 A1 WO2021197051 A1 WO 2021197051A1 CN 2021081056 W CN2021081056 W CN 2021081056W WO 2021197051 A1 WO2021197051 A1 WO 2021197051A1
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tween
hcl
cacl
nacl
buffer
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French (fr)
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贾世香
徐时东
余灵菊
周波
吴洪
袁永龙
李强
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安源医药科技（上海）有限公司
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Priority to US17/907,724 priority Critical patent/US20230134182A1/en
Priority to EP21779289.4A priority patent/EP4130048A4/en
Publication of WO2021197051A1 publication Critical patent/WO2021197051A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/166—Fluid composition conditioning, e.g. gradient
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • B01D15/203—Equilibration or regeneration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B01D15/327—Reversed phase with hydrophobic interaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424—Elution mode
    • B01D15/426—Specific type of solvent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to the field of Fc fusion protein purification; more specifically, it relates to a method for efficiently separating and purifying recombinant human coagulation factor VIII Fc fusion protein.
  • Coagulation factor VIII is a multi-structure macromolecular glycoprotein, divided into 6 domains: three A domains (A1, A2, A3), a carbohydrate-rich and non-essential central domain (B-structure) Domain) and two C domains (C1, C2).
  • the mature protein is composed of a light chain and a heavy chain with a molecular weight of approximately 280 kDa.
  • the molecular weight of the light chain is about 80kDa, the structure includes A3, C1 and C2, and the connection mode is A3-C1-C2.
  • the molecular weight of the heavy chain is about 90 to 200kDa, the structure includes A1, A2 and B, and the connection mode is A1-A2-B.
  • the connection between the heavy chain and the light chain is metal ion dependent.
  • the dimers of heavy and light chains bind von Willebrand (vWF) with high affinity to protect it from degradation before maturation.
  • the purification process of coagulation factor VIII generally has the problems of complex process, high cost, and low purity (Stefan Winge, etc., Protein Expression and Purification, 2015, 115: 165-175).
  • the effective removal of impurities in the affinity chromatography process is a key factor in downstream protein preparation.
  • most of the current FVIII affinity chromatography purification steps have the problems of low recovery rate and high cost.
  • Affinity chromatography is expensive to use in the elution step.
  • Reagents, such as ethylene glycol, propylene glycol, arginine, etc. which shorten the service life of the filler, have more residual organic solvents, reduce the recovery rate, and greatly increase the purification cost.
  • the purpose of the present invention is to establish a method for purifying recombinant human coagulation factor VIII Fc fusion protein with simple operation, high recovery rate and low production cost.
  • the present invention provides a method for separating or purifying recombinant human coagulation factor VIII Fc fusion protein (abbreviated as fusion protein), and the method includes the following steps:
  • Affinity chromatography capture the fusion protein with protein A affinity medium, and then elute the fusion protein with elution buffer to collect the separated products;
  • Anion exchange chromatography load the above-mentioned separation products on a quaternary ammonium salt type strong anion exchange chromatography medium to separate structural variants, aggregates, remove HCP, DNA and other contaminants, and perform gradient washing with elution buffer Remove, collect the separated product;
  • the fusion protein is human FVIII deleted/truncated by natural human FVIII or B domain and its variants are connected to the constant region sequence (Fc domain) of any antibody isotype derived from human via a peptide linker. Its variants are fused; the Fc domain is selected from, for example, human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE heavy chain constant regions or variants thereof; more preferably from human IgG1, IgG2 , IgG3 and IgG4 heavy chain constant regions or variants thereof; in a preferred embodiment of the present invention, the amino acid sequence of the recombinant human coagulation factor VIII Fc fusion protein is shown in SEQ ID NO: 2.
  • the affinity chromatography step is performed on an affinity medium selected from: MabSelect, MabSelect SuRe, Protein A Diamond, and MabSelect SuRe LX; MabSelect SuRe LX is preferably used;
  • a decontamination buffer that does not contain or contains a relatively low concentration of organic solvent is used to rinse to remove some contaminants; the decontamination buffer contains 20mM His-HCl, 0.4-1.5M NaCl, 10mM CaCl 2 , 0.02-0.05% Tween-80, 0-10% ethylene glycol, pH 5.0-7.0; preferably 20mM His-HCl, 1.5M NaCl, 10mM CaCl 2 , 0.02% Tween -80, 3-7% ethylene glycol, pH 5.0-7.0.
  • the elution buffer used in affinity chromatography contains one or more than one salt, one or more than one polyol organic solvent and one or more And the elution buffer further contains calcium ions at a concentration of 5 mM to 20 mM; and the elution buffer further contains 10 mM to 50 mM His-HCL buffer; and the elution buffer
  • the pH of the buffer is 4.0 to 8.0, preferably 5.0 to 6.0, more preferably 5.0;
  • examples of the salt include sodium chloride, ammonium chloride, potassium chloride, sodium sulfate, ammonium sulfate, etc.; the preferred salt in the present invention is chloride; the most preferred salt in the present invention is sodium chloride;
  • the polyhydric alcohol organic solvent includes but is not limited to ethylene glycol, 1,2-propylene glycol and glycerol, preferably ethylene glycol; and the ratio of the organic solvent is 5-40% (w/ w), preferably 5-20%;
  • the calcium ion is selected from soluble calcium salts, including but not limited to calcium chloride, calcium acetate, calcium lactate, calcium benzoate; preferably calcium chloride;
  • the concentration is 0.01% to 0.1% (v/v), preferably 0.02% To 0.05% (v/v).
  • the elution buffer contains 20mM His-HCl, 0.5-2.0M NaCl, 5-20mM CaCl 2 , 0.02-0.1% Tween-80, 2-20% ethylene glycol, and the elution buffer
  • the pH is 4.5 to 7.0;
  • the affinity chromatography specifically includes the following steps:
  • elution buffer (20mM His-HCl, 1.5M NaCl , 10mM CaCl 2, 0.02% Tween-80,15% ethylene glycol, pH 5.0) to give a fusion protein, collecting the target peak.
  • the anion exchange chromatography step is carried out on a strong anion exchange medium selected from the following: Q HP, Toyopearl GigaCap Q-650, DEAE Beads 6FF, Generik MC-Q, Fractogel EMD TMAE, Q Ceramic HyperD F, preferably used Q HP;
  • a strong anion exchange medium selected from the following: Q HP, Toyopearl GigaCap Q-650, DEAE Beads 6FF, Generik MC-Q, Fractogel EMD TMAE, Q Ceramic HyperD F, preferably used Q HP;
  • the anion exchange chromatography specifically includes the following steps:
  • the method for separating or purifying recombinant human coagulation factor VIII Fc fusion protein (abbreviated as fusion protein) provided by the present invention further includes a hydrophobic chromatography step in a flow-through mode, and the specific steps are as follows:
  • Affinity chromatography capture the fusion protein with protein A affinity medium, and then elute the fusion protein with elution buffer to collect the separated products;
  • Anion exchange chromatography load the above separated products on a quaternary ammonium salt strong anion exchange chromatography medium to separate structural variants, remove HCP, DNA and other contaminants, gradient elution, and collect the separated products;
  • the hydrophobic chromatography step in the flow-through mode is before the anion exchange chromatography step, and the specific steps are as follows:
  • Affinity chromatography capture the fusion protein with protein A affinity medium, and then elute the fusion protein with elution buffer to collect the separated products;
  • Anion exchange chromatography load the above separated products on a quaternary ammonium salt strong anion exchange chromatography medium to separate structural variants, remove HCP, DNA and other contaminants, gradient elution, and collect the separated products;
  • the fusion protein is human FVIII deleted/truncated by natural human FVIII or B domain and its variants are connected to the constant region sequence (Fc domain) of any antibody isotype derived from human via a peptide linker. Its variants are fused; the Fc domain is selected from, for example, human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE heavy chain constant regions or variants thereof; more preferably from human IgG1, IgG2 , IgG3 and IgG4 heavy chain constant regions or variants thereof; in a preferred embodiment of the present invention, the amino acid sequence of the recombinant human coagulation factor VIII Fc fusion protein is shown in SEQ ID NO: 2.
  • the affinity chromatography step is performed on an affinity medium selected from: MabSelect, MabSelect SuRe, Protein A Diamond, and MabSelect SuRe LX; MabSelect SuRe LX is preferably used;
  • the elution buffer used in the affinity chromatography step contains one or more than one salt, one or more than one polyol organic solvent and one or more More than one surfactant; and the elution buffer further contains calcium ions at a concentration of 5 mM to 20 mM; and the elution buffer further contains a His-HCL buffer of 10 mM to 50 mM; and the wash
  • the pH of the debuffered solution is 4.0 to 8.0, preferably 5.0 to 6.0, more preferably 5.0;
  • examples of the salt include sodium chloride, ammonium chloride, potassium chloride, sodium sulfate, ammonium sulfate, etc.; the preferred salt in the present invention is chloride; the most preferred salt in the present invention is sodium chloride;
  • the polyhydric alcohol organic solvent includes but is not limited to ethylene glycol, 1,2-propylene glycol and glycerol, preferably ethylene glycol; and the ratio of the organic solvent is 5-40% (w/ w), preferably 5-20%;
  • the calcium ion is selected from soluble calcium salts, including but not limited to calcium chloride, calcium acetate, calcium lactate, calcium benzoate; preferably calcium chloride;
  • the concentration is 0.01% to 0.1% (v/v), preferably 0.02% To 0.05% (v/v).
  • the elution buffer contains 20mM His-HCl, 0.5-2.0M NaCl, 5-20mM CaCl 2 , 0.02% Tween-80, 5-20% ethylene glycol, and the pH of the elution buffer From 5.0 to 7.0;
  • a decontamination buffer that does not contain or contains a relatively low concentration of organic solvent is used to rinse to remove some contaminants; the decontamination buffer contains 20 mM His- HCl, 0.4-1.5M NaCl, 10mM CaCl 2 , 0.02% Tween-80, 0-10% ethylene glycol, pH 5.0-7.0; preferably 20mM His-HCl, 1.5M NaCl, 10mM CaCl 2 , 0.02% Tween -80, 3-7% ethylene glycol, pH 5.0-7.0.
  • the affinity chromatography step specifically includes the following steps:
  • elution buffer (20mM His-HCl, 1.5M NaCl , 10mM CaCl 2, 0.02% Tween-80,15% ethylene glycol, pH 5.0) to give a fusion protein, collecting the target peak.
  • the hydrophobic chromatography step is performed on a weakly hydrophobic medium selected from the following: Capto Octyl, Octyl Sepharose 4FF, Phenyl Sepharose 6FF; preferably Capto Octyl is used;
  • the equilibration buffer used in the step of hydrophobic chromatography comprises 20mM His-HCl, 0.1-1.0M NaCl, 10mM CaCl 2, 0.02% Tween-80,0-10% ethylene glycol, pH 6.8 to 7.2.
  • the hydrophobic chromatography step specifically includes the following steps:
  • the anion exchange chromatography step is performed on a strong anion exchange medium selected from the group consisting of Q HP, Toyopearl GigaCap Q-650, DEAE Beads 6FF, Generik MC-Q, Fractogel EMD TMAE, Q Ceramic HyperD F, preferably Use Q HP locally;
  • the anion exchange chromatography step specifically includes the following steps:
  • the purification production cost is greatly reduced, the use of most expensive reagents (for example, arginine) is removed, and the type and amount of organic solvents used are greatly reduced, so that the residual amount of organic reagents is greatly reduced, which not only reduces
  • the detection step also greatly increases the number of repeated uses of the filler, and it is easier to meet the limit requirements for organic solvent residues in drug registration;
  • HCP host cell protein
  • product-related impurities for example, high molecular weight (HMW) and low molecular weight (LMW) substances are key factors affecting the downstream protein purification process.
  • HMW high molecular weight
  • LMW low molecular weight
  • Protein purity significantly affects the number and type of subsequent purification steps.
  • the method provided by the present invention is purified by affinity chromatography in one step, the protein purity can reach over 85%, which greatly reduces the polymer content, which greatly reduces the difficulty of downstream purification and simplifies the subsequent purification process, for example, the molecular sieve layer can be removed Analyzing steps, so that the total recovery rate is greatly improved;
  • the hydrophobic chromatography step is changed from adsorption elution mode to flow-through mode, which can greatly reduce the amount of resin used; the purity can be increased to about 95%, and the recovery rate can reach 60-80%;
  • the process has no pollution, no waste water, and is more environmentally friendly
  • the purity of the purified sample can reach more than 95%, and the overall recovery rate can reach about 25%, which reduces the production cost and is easy to scale up.
  • Fractor VIII or “FVIII” as used herein refers to a human plasma glycoprotein that is a member of the endogenous coagulation pathway and is essential for coagulation.
  • Natural FVIII is a full-length human FVIII molecule shown in SEQ ID NO:1 (amino acids 1-2332), and its B domain corresponds to the sequence shown in SEQ ID NO:1 with amino acid numbers 741-1648.
  • the Factor VIII molecule of the present invention may be a B-domain deleted/truncated FVIII molecule and variants thereof, wherein the remaining domains basically correspond to the sequences shown in amino acid numbers 1-740 and 1649-2332 in SEQ ID NO:1.
  • the B-domain deleted/truncated FVIII molecule of the present invention is a variant with amino acid modifications (substitutions, additions or deletions, etc.) introduced at certain positions, meaning that the remaining domains (ie 3 A structures) Domain and 2 C domains) corresponding to SEQ ID NO:1 (amino acids 1-740 and 1649-2332) may have, for example, 1-20 or more amino acid substitutions, deletions or additions; or have approximately 1%, 2%, 3%, 4%, 5%, 10%, 15% or 20% difference; the above-mentioned modified B-domain deleted/truncated Factor VIII variant has FVIII activity, which is natural human At least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and 100% or even more than 100% of FVIII activity.
  • Fc fusion protein or “Fc fusion derivative” is meant herein to include FVIII derivatives of the present invention fused to an Fc domain that can be derived from any antibody isotype.
  • the Fc domain may also be modified to modulate certain effector functions, such as complement fixation and/or binding to certain Fc receptors.
  • the fusion of FVIII with the Fc domain that has the ability to bind to the FcRn receptor will generally lead to an extension of the circulating half-life of the fusion protein (compared to the half-life of natural FVIII).
  • the term "elution buffer” refers to its conventional meaning, and refers to a buffer or buffer with a suitable pH and/or ionic strength to release one or more proteins from the separation matrix.
  • the "salt" in the elution step refers to alkaline earth metal, alkali metal salt or ammonium salt, that is, a salt having an inorganic or organic cation derived from an alkaline earth metal or an alkali metal element or an ammonium cation.
  • Examples of such salts include sodium chloride, ammonium chloride, sodium sulfate, ammonium sulfate, and the like.
  • the preferred salt in the present invention is chloride or sulfate; the most preferred salt in the present invention is sodium chloride.
  • the term "buffering agent” includes those agents capable of maintaining the pH of the solution within a desired range. In the context of the present invention, it is in the range of 4.0 to 8.0, for example 5.0.
  • the buffer concentration range is selected to maintain the preferred pH of the solution.
  • the buffer can also be a mixture of at least two buffers, where the mixture can provide a pH within a specified range.
  • the buffer may include, but is not limited to, histidine (L-histidine), TRIS. In one embodiment, the buffer used is 20 mM histidine.
  • Example 1 Purification of recombinant human coagulation factor VIII Fc fusion protein by two-step chromatography
  • the isolated or purified recombinant human coagulation factor VIII Fc fusion protein described in this example has an amino acid sequence as shown in SEQ ID NO: 2 in the sequence table of this application, that is, as described in CN 201811123918.X as shown in SEQ ID NO: 9
  • the fusion protein shown is expressed by Chinese hamster ovary (CHO) cells, and the construction method of the expression vector refers to the international application CN 201811123918.X.
  • the fermentation broth obtained by the above method and the supernatant obtained after centrifugation or deep filtration are used for subsequent separation or purification of the fusion protein.
  • a two-step chromatography method was used to purify the recombinant human coagulation factor VIII protein.
  • They are affinity chromatography and anion exchange chromatography (the protein purifier used in this example is AKTA pure 25M from GE, USA.
  • the reagents used in this example are all purchased from Sinopharm Chemical Reagent Co., Ltd., and the purity is analysis class).
  • the first step, affinity chromatography use GE's Mabselect Sure or other commercially available affinity chromatography media for sample capture, concentration, and removal of some contaminants.
  • the second step, anion exchange chromatography use Boglung's Q-HP or other commercially available anion exchange chromatography media for purification to separate structural variants and further remove HCP, DNA and other contaminants.
  • First use equilibration buffer 20mM His-HCl, 100mM NaCl, 10mM CaCl 2 , 0.02% Tween-80, pH 7.0-7.5, flush the column with a linear flow rate of 50-200cm/h for 3-5 column volumes (CV ); The load is controlled at 5000-50000IU/ml; before loading, the sample is diluted 3-10 times with diluent (20mM His-HCl, 10mM NaCl, 10mM CaCl 2 , 0.02% Tween-80, pH 7.0), After loading the sample, use the equilibration buffer: 20mM His-HCl, 100mM NaCl, 10mM CaCl 2 , 0.02% Tween-80, pH 7.0-7.5, and wash 3-5 columns at a linear
  • Elution buffer 20mM His-HCl, 1M NaCl, 10mM CaCl 2 , 0.02% Tween-80, pH 7.0- 7.5.
  • the condition is that the elution buffer is from 50%-70%, elution is 3-5 column volumes (CV), and the linear flow rate is controlled at no higher than 200cm/h.
  • the eluted samples are collected, and the samples are sent for protein content, SEC-HPLC, activity and HCP content detection. The purity can reach 95%, and the recovery rate is 70%. The remaining samples were immediately exchanged with G25 or UF/DF to the preparation solution for storage.
  • Example 2 Purification of recombinant human coagulation factor VIII Fc fusion protein by three-step chromatography
  • the source of the isolated or purified recombinant human coagulation factor VIII Fc fusion protein described in this example is the same as in Example 1.
  • This example uses a three-step chromatography method to purify the recombinant human coagulation factor VIII Fc fusion protein, which are affinity chromatography, hydrophobic chromatography, and anion exchange chromatography (the protein purifier used in this example is from GE, USA AKTA pure 25M; the reagents used in this example are all purchased from Sinopharm Chemical Reagent Co., Ltd., and the purity is all analytical grade).
  • the first step, affinity chromatography use GE's Mabselect Sure or other commercially available affinity chromatography media for sample capture, concentration, and removal of some contaminants.
  • the second step, hydrophobic chromatography use GE's Capto Octyl or other commercially available hydrophobic chromatography media for intermediate purification to reduce the polymer content.
  • the goal is to partially remove the polymer to make the content less than 5%.
  • the affinity sample is diluted 2 times with diluent 20mM His-HCl, 10mM NaCl, 10mM CaCl 2 , 0.02% Tween-80, pH 7.0, and equilibration buffer: 20mM His-HCl, 0.1-1M NaCl, 10mM CaCl 2 , 0-8% ethylene glycol, 0.02% Tween-80, pH 6.8-7.2, equilibrate the column with a linear flow rate of 50-200cm/h for 3-5 column volumes (CV); the capacity is controlled at ⁇ 100000IU/ ml; After loading the sample, flush the column with 3-5 column volumes (CV) at a linear flow rate of 50-200cm/h with the equilibration buffer
  • the third step, anion exchange chromatography use Boglung's Q-HP or other commercially available anion exchange chromatography media for purification to separate structural variants and further remove contaminants such as HCP and DNA.
  • First use equilibration buffer 20mM His-HCl, 100mM NaCl, 10mM CaCl 2 , 0.02% Tween-80, pH 7.0-7.5, and flush the column with a linear flow rate of 50-200cm/h for 3-5 column volumes (CV ); The load is controlled at 5000-10000IU/ml; before loading, the sample is diluted 0-3 times with diluent (20mM His-HCl, 10mM NaCl, 10mM CaCl 2 , 0.02% Tween-80, pH 7.0),
  • Use equilibration buffer 20mM His-HCl, 100mM NaCl, 10mM CaCl 2 , 0.02% Tween-80, pH 7.0-7.5, flush the column with a linear flow rate of 50-
  • the above-mentioned anion exchange chromatography step is followed by the affinity chromatography step, and finally the hydrophobic chromatography step in the flow-through mode is performed.

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Abstract

公开了一种高效分离纯化重组人凝血VIII因子Fc融合蛋白的方法。该方法包括亲和层析以及阴离子交换层析步骤；并且该亲和层析捕获样品在pH 4.0至8.0条件下，采用含5-20％多元醇类有机溶剂的盐离子缓冲液进行洗脱，蛋白样品经一步ProteinA亲和层析分离即可提纯至85％以上；所述纯化方法操作简便，每步层析之间自然衔接，回收率高、成本低且容易放大生产。

Description

高效分离纯化重组人凝血因子VIII Fc融合蛋白的方法 技术领域
本发明涉及Fc融合蛋白纯化领域；更具体地，涉及一种高效分离纯化重组人凝血因子VIII Fc融合蛋白的方法。
背景技术
凝血因子VIII(FVIII)是多结构的大分子糖蛋白，分为6个结构域：三个A结构域(A1、A2、A3)，一个富碳水化合物且非必需的中央结构域(B-结构域)和两个C结构域(C1、C2)。成熟的蛋白质由大约分子量为280kDa的轻链和重链组成。轻链分子量约为80kDa，结构包括A3，C1和C2，连接方式为A3-C1-C2。重链分子量约90至200kDa，结构包括A1，A2和B，连接方式为A1-A2-B。重链和轻链连接是金属离子依赖性的。在血浆中，重链和轻链的二聚体再以高亲和力结合冯.维勒布兰德因子(von Willebrand,vWF)，保护其免于成熟前降解。
目前，凝血因子VIII的纯化工艺普遍存在工艺复杂、成本高、纯度低的问题(Stefan Winge等,Protein Expression and Purification,2015,115:165-175)。亲和层析过程有效去除杂质是下游蛋白制备中的关键因素，然而目前FVIII亲和层析纯化步骤多存在回收率低和成本较高的问题，亲和层析在洗脱步骤中使用大量昂贵试剂，如乙二醇、丙二醇、精氨酸等，这使得填料的使用寿命缩短，有机溶剂残留较多，回收率降低，且大大增加了纯化成本。虽然GE公司开发出新型纯化填料VIII select(Justin T.McCue等,Journal of chromatography A,2009,1216:7824-7830)，但仍存在填料成本较高、回收率低以及洗脱液中使用的试剂昂贵等问题。目前已申报IND或已上市的长效人凝血VIII因子的药物生产厂家，例如，辅仁、Biogen等开发的重组人凝血因子VIII长效产品的纯化工艺依旧存在操作繁琐、成本高及回收率低等问题。
全球现有大量罹患血友病A患者，还有许多未被诊断或登记，市场需求远远大于产能，而重组人凝血因子VIII Fc融合蛋白的高价格使得药物无法普及应用，而生产成本又是重组蛋白药物价格一直居高不下的主要因素。因此，本领域亟待开发一种高效便捷、成本低廉且适于工业应用的纯化重组人凝血因子VIII Fc融合蛋白的新方法。
发明内容：
本发明的目的在于建立一种操作简便、回收率高、生产成本低的纯化重组人凝血因子VIII  Fc融合蛋白方法。
本发明提供一种分离或纯化重组人凝血因子VIII Fc融合蛋白(简称融合蛋白)的方法，该方法包括以下步骤：
(1)亲和层析：用蛋白A亲和介质捕获融合蛋白，再以洗脱缓冲液洗脱融合蛋白，收集分离产物；
(2)阴离子交换层析：将上述分离产物加载于季铵盐类强阴离子交换层析介质上以分离结构变异体、聚体、去除HCP、DNA等污染物，以洗脱缓冲液进行梯度洗脱，收集分离产物；
其中，所述融合蛋白是由天然人FVIII或B结构域缺失的/截短的人FVIII及其变体通过肽接头与来源于人的任何抗体同种型的恒定区序列(Fc结构域)或其变体融合而成；所述Fc结构域选自例如人IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区或其变体；更优选自人IgG1、IgG2、IgG3和IgG4的重链恒定区或其变体；本发明的一优选实施例中，所述重组人凝血因子VIII Fc融合蛋白的氨基酸序列如SEQ ID NO：2所示。
优选地，亲和层析步骤在选自下列的亲和介质上进行：MabSelect、MabSelect SuRe、Protein A Diamond和MabSelect SuRe LX；优选地使用MabSelect SuRe LX；
优选地，亲和层析中融合蛋白洗脱前先使用不含或含有较低浓度的有机溶剂的去污缓冲液淋洗以去除部分污染物；所述去污缓冲液包含20mM His-HCl、0.4-1.5M NaCl、10mM CaCl 2、0.02-0.05％Tween-80、0-10％乙二醇，pH为5.0-7.0；优选为20mM His-HCl、1.5M NaCl、10mM CaCl 2、0.02％Tween-80、3-7％乙二醇，pH为5.0-7.0。
其中，亲和层析中使用的洗脱缓冲液包含浓度为0.2M至1.5M的一种或多于一种的盐、一种或多于一种的多元醇类有机溶剂和一种或多于一种的表面活性剂；和所述洗脱缓冲液还含有浓度为5mM至20mM的钙离子；和所述洗脱缓冲液还含有10mM至50mM的His-HCL缓冲剂；和所述洗脱缓冲液的pH为4.0至8.0，优选为5.0至6.0，更优选为5.0；
优选地，其中所述盐的实例包括氯化钠、氯化铵、氯化钾、硫酸钠、硫酸铵等；本发明优选的盐是氯化物；本发明中最优选的盐是氯化钠；
优选地，其中所述多元醇类有机溶剂包括但不限于乙二醇、1,2-丙二醇和丙三醇，优选为乙二醇；并且所述有机溶剂的比例为5-40％(w/w)，优选为5-20％；
优选地，其中所述钙离子选自可溶性钙盐，包括但不限于氯化钙、乙酸钙、乳酸钙、苯甲酸钙；优选为氯化钙；
优选地，其中所述表面活性剂为吐温-80(Tween-80)或曲拉通X-100(Triton X-100)，浓度为0.01％至0.1％(v/v)，优选为0.02％至0.05％(v/v)。
优选地，所述洗脱缓冲液包含20mM His-HCl、0.5-2.0M NaCl、5-20mM CaCl 2、0.02-0.1％Tween-80、2-20％乙二醇，和所述洗脱缓冲液的pH为4.5至7.0；
本发明的一优选实施例中，所述亲和层析具体包括下列步骤：
(1)用平衡缓冲液(20mM His-HCl、0.2M NaCl、10mM CaCl 2、0.02％Tween-80、pH为6.8-7.2)平衡层析柱3-5个柱体积；
(2)上样，载量不高于50000IU/ml；
(3)完成上样后，用上述平衡缓冲液再平衡3-5个柱体积，冲洗未结合的组分；
(4)使用去污缓冲液(20mM His-HCl、0.5M NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0)淋洗层析柱3-5个柱体积，以去除部分污染物；
(5)最后用洗脱缓冲液(20mM His-HCl、1.5M NaCl、10mM CaCl 2、0.02％Tween-80、15％乙二醇，pH为5.0)洗脱融合蛋白，收集目标峰。
优选地，阴离子交换层析步骤在选自下列的强阴离子交换介质上进行：Q HP、Toyopearl GigaCap Q-650、DEAE Beads 6FF、Generik MC-Q、Fractogel EMD TMAE、Q Ceramic HyperD F，优选地使用Q HP；
本发明的一优选实施例中，所述阴离子交换层析具体包括下列步骤：
(1)用稀释液(20mM His-HCl、10mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0)将亲和洗脱产物进行稀释3～10倍；
(2)再用平衡缓冲液(20mM His-HCl、100mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)平衡层析柱3-5个柱体积；
(3)上样，上样载量在5000-50000IU/ml；
(4)上样结束后，用上述平衡缓冲液冲洗层析柱3-5个柱体积；
(5)用洗脱缓冲液(20mM His-HCl、500mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)冲洗层析柱3-5个柱体积，以去除部分杂蛋白；
(6)最后用层级梯度的盐离子洗脱缓冲液(20mM His-HCl、1M NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)进行洗脱，条件为洗脱缓冲液从50％至70％，洗脱3-5个柱体积，收集洗脱样品。
优选地，本发明提供的所述分离或纯化重组人凝血因子VIII Fc融合蛋白(简称融合蛋白)的方法还包括流穿模式的疏水层析步骤，具体步骤如下：
(1)亲和层析：用蛋白A亲和介质捕获融合蛋白，再以洗脱缓冲液洗脱融合蛋白，收集分离产物；
(2)阴离子交换层析：将上述分离产物加载于季铵盐类强阴离子交换层析介质上以分离结构变异体、去除HCP、DNA等污染物，梯度洗脱，收集分离产物；
(3)疏水层析：将上述分离产物加载于含四碳或八碳的弱疏水性介质上，然后用平衡缓冲液淋洗层析柱，使上述分离产物中的杂质及聚合体结合到疏水层析介质上，融合蛋白直接流穿，收集分离产物；
或，所述流穿模式的疏水层析步骤在所述阴离子交换层析步骤之前，具体步如下：
(1)亲和层析：用蛋白A亲和介质捕获融合蛋白，再以洗脱缓冲液洗脱融合蛋白，收集分离产物；
(2)疏水层析：将上述分离产物加载于含四碳或八碳的弱疏水性介质上，然后用平衡缓冲液淋洗层析柱，使上述分离产物中的杂质及聚合体结合到疏水层析介质上，融合蛋白直接流穿，收集分离产物；
(3)阴离子交换层析：将上述分离产物加载于季铵盐类强阴离子交换层析介质上以分离结构变异体、去除HCP、DNA等污染物，梯度洗脱，收集分离产物；
其中，所述融合蛋白是由天然人FVIII或B结构域缺失的/截短的人FVIII及其变体通过肽接头与来源于人的任何抗体同种型的恒定区序列(Fc结构域)或其变体融合而成；所述Fc结构域选自例如人IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区或其变体；更优选自人IgG1、IgG2、IgG3和IgG4的重链恒定区或其变体；本发明的一优选实施例中，所述重组人凝血因子VIII Fc融合蛋白的氨基酸序列如SEQ ID NO：2所示。
优选地，亲和层析步骤在选自下列的亲和介质上进行：MabSelect、MabSelect SuRe、Protein A Diamond和MabSelect SuRe LX；优选地使用MabSelect SuRe LX；
其中，亲和层析步骤中使用的洗脱缓冲液包含浓度为0.2M至1.5M的一种或多于一种的盐、一种或多于一种的多元醇类有机溶剂和一种或多于一种的表面活性剂；和所述洗脱缓冲液还含有浓度为5mM至20mM的钙离子；和所述洗脱缓冲液还含有10mM至50mM的His-HCL缓冲剂；和所述洗脱缓冲液的pH为4.0至8.0，优选为5.0至6.0，更优选为5.0；
优选地，其中所述盐的实例包括氯化钠、氯化铵、氯化钾、硫酸钠、硫酸铵等；本发明优选的盐是氯化物；本发明中最优选的盐是氯化钠；
优选地，其中所述多元醇类有机溶剂包括但不限于乙二醇、1,2-丙二醇和丙三醇，优选为乙二醇；并且所述有机溶剂的比例为5-40％(w/w)，优选为5-20％；
优选地，其中所述钙离子选自可溶性钙盐，包括但不限于氯化钙、乙酸钙、乳酸钙、苯甲酸钙；优选为氯化钙；
优选地，其中所述表面活性剂为吐温-80(Tween-80)或曲拉通X-100(Triton X-100)，浓度为0.01％至0.1％(v/v)，优选为0.02％至0.05％(v/v)。
优选地，所述洗脱缓冲液包含20mM His-HCl、0.5-2.0M NaCl、5-20mM CaCl 2、0.02％Tween-80、5-20％乙二醇，和所述洗脱缓冲液的pH为5.0至7.0；
优选地，亲和层析步骤中，融合蛋白洗脱前先使用不含或含有较低浓度的有机溶剂的去污缓冲液淋洗以去除部分污染物；所述去污缓冲液包含20mM His-HCl、0.4-1.5M NaCl、10mM CaCl 2、0.02％Tween-80、0-10％乙二醇，pH为5.0-7.0；优选为20mM His-HCl、1.5M NaCl、10mM CaCl 2、0.02％Tween-80、3-7％乙二醇，pH为5.0-7.0。
本发明的一优选实施例中，亲和层析步骤具体包括下列步骤：
(1)用平衡缓冲液(20mM His-HCl、0.2M NaCl、10mM CaCl 2、0.02％Tween-80、pH为6.8-7.2)平衡层析柱3-5个柱体积；
(2)上样，载量不高于50000IU/ml；
(3)完成上样后，用上述平衡缓冲液再平衡3-5个柱体积，冲洗未结合的组分；
(4)使用去污缓冲液(20mM His-HCl、0.5M NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0)淋洗层析柱3-5个柱体积，以去除部分污染物；
(5)最后用洗脱缓冲液(20mM His-HCl、1.5M NaCl、10mM CaCl 2、0.02％Tween-80、15％乙二醇，pH为5.0)洗脱融合蛋白，收集目标峰。
优选地，所述疏水层析步骤在选自下列的弱疏水性介质上进行：Capto Octyl、Octyl Sepharose 4FF、Phenyl Sepharose 6FF；优选地使用Capto Octyl；
其中，所述疏水层析步骤中使用的平衡缓冲液包含20mM His-HCl、0.1-1.0M NaCl、10mM CaCl 2、0.02％Tween-80、0-10％乙二醇，pH为6.8-7.2。
本发明的一优选实施例中，所述疏水层析步骤具体包括下列步骤：
(1)用稀释液(20mM His-HCl、10mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0)将亲和洗脱产物进行稀释；
(2)再用平衡缓冲液(20mM His-HCl、0.1-1M NaCl、10mM CaCl 2、0.02％Tween-80、0-8％乙二醇，pH为6.8-7.2)平衡层析柱3-5个柱体积；
(3)上样，上样载量小于100000IU/ml；
(4)上样结束后，用上述平衡缓冲液冲洗层析柱3-5个柱体积，收集流穿产物。
优选地，所述阴离子交换层析步骤在选自下列的强阴离子交换介质上进行：Q HP、Toyopearl GigaCap Q-650、DEAE Beads 6FF、Generik MC-Q、Fractogel EMD TMAE、Q Ceramic HyperD F，优选地使用Q HP；
本发明的一优选实施例中，所述阴离子交换层析步骤具体包括下列步骤：
(1)用稀释液(20mM His-HCl、10mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0)将疏水层析流穿产物进行稀释；
(2)再用平衡缓冲液(20mM His-HCl、100mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)平衡层析柱3-5个柱体积；
(3)上样，上样载量在5000-10000IU/ml；
(4)上样结束后，用上述平衡缓冲液冲洗层析柱3-5个柱体积；
(5)用洗脱缓冲液(20mM His-HCl、500mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)冲洗层析柱3-5个柱体积，以去除部分杂蛋白；
(6)最后用层级梯度的盐离子洗脱缓冲液(20mM His-HCl、1M NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)进行洗脱，条件为洗脱缓冲液从50％至70％，洗脱3-5个柱体积，收集洗脱样品。
本发明提供的分离或纯化重组人凝血因子VIII Fc融合蛋白的方法具有如下的优点：
1、纯化生产成本极大的降低，去除掉大部分昂贵试剂(例如，精氨酸)的使用，并大幅减少有机溶剂的使用种类和用量，使得有机试剂的残留量大幅降低，这不仅减少了检测步骤，也使得填料的重复使用次数大幅提升，也更易满足药品注册中关于有机溶剂残留的限度要求；
2、在亲和层析步骤有效去除宿主细胞蛋白(HCP)和产物相关杂质，如高分子量(HMW)和低分子量(LMW)物质是影响下游蛋白纯化工艺的关键因素，“捕获步骤”后的蛋白纯度显著影响后续纯化步骤的数目和类型。本发明提供的方法经亲和层析一步纯化后，蛋白纯度即可达到85％以上，使聚体含量大幅降低，这大大降低了下游纯化的难度，简化了后续纯化工艺，例如可以去除分子筛层析步骤，使总回收率大幅提高；
3、疏水层析步骤由吸附洗脱模式改为流穿模式，可大大减少树脂使用量；纯度可提升至95％左右，回收率可达60-80％；
4、不同纯化步骤间样品处理少，工艺操作简便，步骤之间自然衔接，易于工业化；例如， 疏水层析流穿样品无需任何处理，可直接上样进行阴离子交换层析；
5、工艺过程无污染、无废水，对环境更友好；
6、纯化样品纯度能达到95％以上，总体回收率达到25％左右，使得生产成本降低，易于工艺放大。
附图说明
图1、亲和层析洗脱样品SEC-HPLC检测结果
图2、疏水层析流穿样品SEC-HPLC检测结果
图3、离子交换层析洗脱样品SEC-HPLC检测结果
图4、纯化样品换液后SEC-HPLC检测结果
定义
本文所用的“因子VIII”或“FVIII”是指作为内源性凝血途径的成员并对凝血而言至关重要的人血浆糖蛋白。“天然FVIII”是如SEQ ID NO：1(氨基酸1-2332)所示的全长人FVIII分子，其B结构域对应SEQ ID NO：1中氨基酸编号741-1648所示序列。
本发明的因子VIII分子可以是B结构域缺失的/截短的FVIII分子及其变体，其中剩余结构域基本对应于SEQ ID NO：1中氨基酸编号1-740和1649-2332所示序列。在某些方面，本发明的B结构域缺失的/截短的FVIII分子是在某些位置引入氨基酸修饰(取代、添加或缺失等)的变体，意即剩余结构域(即3个A结构域和2个C结构域)与SEQ ID NO：1对应的氨基酸序列(氨基酸1-740和1649-2332)可具有例如1～20个或更多个氨基酸的取代、缺失或添加；或具有约1％、2％、3％、4％、5％、10％、15％或20％的差异；上述修饰的B结构域缺失的/截短的因子VIII变体具有FVIII活性，其为天然人FVIII活性的至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、和100％或甚至超过100％。
“Fc融合蛋白”或“Fc融合衍生物”在本文中意指包括与可来源于任何抗体同种型的Fc结构域融合的本发明FVIII衍生物。Fc结构域还可经修饰以调节某些效应子功能，例如补体结合和/或与某些Fc受体结合。FVIII与具有与FcRn受体结合的能力的Fc结构域的融合，通常将会导致融合蛋白的循环半衰期延长(与天然FVIII的半衰期相比)。
术语“洗脱缓冲液”是指其常规含义，是指具有合适pH和/或离子强度的缓冲剂或缓冲剂，以从分离基质中释放一种或多种蛋白质。如本文所用，洗脱步骤中的“盐”是指碱土金属，碱金属盐或铵盐，即具有来自碱土金属或碱金属元素或铵阳离子并具有无机或有机阳离 子的盐。这类盐的实例包括氯化钠、氯化铵、硫酸钠、硫酸铵等。本发明优选的盐是氯化物或硫酸盐；本发明中最优选的盐是氯化钠。
术语“缓冲剂”包括能够将溶液的pH维持在所需范围内的那些试剂。在本发明的上下文中，在4.0至8.0的范围内，例如5.0。选择缓冲液浓度范围以保持溶液的优选pH。缓冲剂也可以是至少两种缓冲剂的混合物，其中混合物能够提供指定范围内的pH值。缓冲剂可包括但不限于组氨酸(L-组氨酸)、TRIS。在一个实施方案中，使用的缓冲剂是20mM组氨酸。
具体实施方式
为了便于理解，以下将通过具体的附图和实施例对本发明进行详细地描述。需要特别指出的是，这些描述仅仅是示例性的描述，并不构成对本发明范围的限制。依据本说明书的论述，本发明的许多变化、改变对所属领域技术人员来说都是显而易见的。
实施例1、两步层析法纯化重组人凝血因子VIII Fc融合蛋白
本实施例中描述的被分离或纯化的重组人凝血因子VIII Fc融合蛋白其氨基酸序列如本申请序列表中SEQ ID NO：2所示，即CN 201811123918.X中记载的如SEQ ID NO：9所示的融合蛋白，由中国仓鼠卵巢(CHO)细胞表达，表达载体构建方法参照国际申请CN 201811123918.X。上述方法获得的发酵液，经离心或深层过滤后得到的上清液用于后续融合蛋白的分离或纯化。
本实施例采用两步层析法对该重组人凝血因子VIII蛋白进行纯化。分别为亲和层析和阴离子交换层析(本实施例采用的蛋白纯化仪为美国GE公司的AKTA pure 25M。本实施例中采用的试剂均购自国药集团化学试剂有限公司，纯度均为分析级)。
第一步，亲和层析：采用GE公司的Mabselect Sure或其它市售的亲和层析介质进行样品捕获、浓缩以及部分污染物的去除。首先使用平衡缓冲液：20mM His-HCl、0.2M NaCl、10mM CaCl 2、0.02％Tween-80，pH 6.8-7.2；以50-200cm/h的线性流速平衡层析柱3-5个柱体积(CV)；将经过澄清后的发酵液以50-200cm/h的线性流速上样，载量不高于50000IU/ml；上样完毕后，使用平衡缓冲液：20mM His-HCl、0.2M NaCl、10mM CaCl 2、0.02％Tween-80，pH 6.8-7.2；以50-200cm/h的线性流速平衡层析柱3-5个柱体积(CV)，冲洗未结合的组份；使用去污缓冲液：20mM His-HCl、0.5M NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0，以50-200cm/h的线性流速冲洗层析柱3-5个柱体积，去除部分污染物；使用洗脱缓冲液：20mM His-HCl、1.5M NaCl、10Mm CaCl 2、0.02％Tween-80，15％乙二醇，pH 5.0，以50-200cm/h的线性流速洗脱目标产物，收集目标峰，检测样品纯度达90％，回收率达75％。
第二步，阴离子交换层析：使用博格隆公司的Q-HP或其它市售的阴离子交换层析介质精纯，分离结构变异体、进一步去除HCP、DNA等污染物。首先使用平衡缓冲液：20mM His-HCl、100mM NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0-7.5，以50-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；载量控制在5000-50000IU/ml；上样前，先将样品用稀释液(20mM His-HCl、10mM NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0)稀释3-10倍，上样完毕后使用平衡缓冲液：20mM His-HCl、100mM NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0-7.5，以50-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；之后使用洗脱缓冲液：20mM His-HCl、500mM NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0-7.5，以50-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)，去除部分杂蛋白，之后采用层级梯度的盐浓度进行洗脱，洗脱缓冲液：20mM His-HCl、1M NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0-7.5，条件为洗脱缓冲液从50％-70％，洗脱3-5个柱体积(CV)，线性流速控制在不高于200cm/h，收集洗脱样品，分别送样进行蛋白含量、SEC-HPLC、活性和HCP含量检测。纯度可达95％，回收率为70％。剩余样品立即使用G25或者UF/DF换液至制剂溶液中保存。
实施例2、三步层析法纯化重组人凝血因子VIII Fc融合蛋白
本实施例中描述的被分离或纯化的重组人凝血因子VIII Fc融合蛋白来源同实施例1。本实施例采用三步层析法对该重组人凝血因子VIII Fc融合蛋白进行纯化，分别为亲和层析、疏水层析、阴离子交换层析(本实施例采用的蛋白纯化仪为美国GE公司的AKTA pure 25M；本实施例中采用的试剂均购自国药集团化学试剂有限公司，纯度均为分析级)。
第一步，亲和层析：采用GE公司的Mabselect Sure或其它市售的亲和层析介质进行样品捕获、浓缩以及部分污染物的去除。首先使用平衡缓冲液：20mM His-HCl、0.2M NaCl、10mM CaCl 2、0.02％Tween-80，pH 6.8-7.2；以50-200cm/h的线性流速平衡层析柱3-5个柱体积(CV)；将经过澄清后的发酵液以50-200cm/h的线性流速上样，载量不高于50000IU/ml；上样完毕后，使用平衡缓冲液：20mM His-HCl、0.2M NaCl、10mM CaCl 2、0.02％Tween-80，pH 6.8-7.2；以50-200cm/h的线性流速平衡层析柱3-5个柱体积(CV)，冲洗未结合的组份；使用去污缓冲液：20mM His-HCl、0.5M NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0，以50-200cm/h的线性流速冲洗层析柱3-5个柱体积，去除部分污染物；使用洗脱缓冲液：20mM His-HCl、1.5M NaCl、10Mm CaCl 2、0.02％Tween-80、15％乙二醇，pH 5.0，以50-200cm/h的线性流速洗脱目标产物，收集目标峰，并进行检测。亲和层析洗脱产物的SEC-HPLC检测结果见图1，检测样品纯度可达88％，回收率达70％。
第二步，疏水层析：使用GE公司的Capto Octyl或其它市售的疏水层析介质进行中间纯化，用于降低聚合体含量，目标是部分去除聚合体，使其含量低于5％。首先，亲和样品使用稀释液20mM His-HCl、10mM NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0稀释2倍，使用平衡缓冲液：20mM His-HCl、0.1-1M NaCl、10mM CaCl 2、0-8％乙二醇，0.02％Tween-80，pH 6.8-7.2，以50-200cm/h的线性流速平衡层析柱3-5个柱体积(CV)；载量控制在<100000IU/ml；上样完毕后，使用平衡缓冲液以50-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)，收集样品分别送检SEC-HPLC，活性及电泳。疏水层析流穿产物的SEC-HPLC检测结果见图2，纯度可达94％，回收率为85％。
第三步，阴离子交换层析：使用博格隆公司的Q-HP或其它市售的阴离子交换层析介质精纯，分离结构变异体、进一步去除HCP、DNA等污染物。首先使用平衡缓冲液：20mM His-HCl、100mM NaCl、10mM CaCl 2、0.02％Tween-80、pH 7.0-7.5，以50-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；载量控制在5000-10000IU/ml；上样前，先将样品用稀释液(20mM His-HCl、10mM NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0)稀释0-3倍，使用平衡缓冲液：20mM His-HCl、100mM NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0-7.5，以50-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；之后使用洗脱缓冲液：20mM His-HCl、500mM NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0-7.5，以50-200cm/h的线性流速冲洗层析柱3-5个柱体积(CV)，去除部分杂蛋白，之后采用层级梯度的盐浓度进行洗脱，洗脱缓冲液：20mM His-HCl、1M NaCl、10mM CaCl 2、0.02％Tween-80，pH 7.0-7.5，条件为洗脱缓冲液从50％-70％，洗脱3-5个柱体积(CV)，线性流速控制在不高于200cm/h，收集洗脱样品，分别送样进行蛋白含量、SEC-HPLC、活性和HCP含量检测。阴离子交换层析洗脱产物的SEC-HPLC检测结果见图3，纯度可达95％，回收率为70％。剩余样品立即使用G25或者UF/DF换液至制剂溶液中保存，换液后产物的SEC-HPLC检测结果见图4。
在某些实施方式中，上述阴离子交换层析步骤在亲和层析步骤之后，最后进行流穿模式的疏水层析步骤。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (17)

  1. 一种分离或纯化重组人凝血因子VIII Fc融合蛋白的方法，所述方法包括以下步骤：
    (1)亲和层析：用蛋白A亲和介质捕获融合蛋白，再以洗脱缓冲液洗脱融合蛋白，收集分离产物；
    (2)阴离子交换层析：将上述步骤分离产物加载于季铵盐类强阴离子交换层析介质上，以洗脱缓冲液进行梯度洗脱，收集分离产物；
    其中，所述融合蛋白是由天然人FVIII或B结构域缺失的/截短的人FVIII及其变体通过肽接头与来源于人的任何抗体同种型的恒定区序列(Fc结构域)或其变体融合而成；所述Fc结构域选自例如人IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区或其变体；更优选自人IgG1、IgG2、IgG3和IgG4的重链恒定区或其变体；本发明的一优选实施例中，所述重组人凝血因子VIII Fc融合蛋白的氨基酸序列如SEQ ID NO：2所示；
    其中，亲和层析步骤中使用的洗脱缓冲液包含浓度为0.2M至1.5M的一种或多于一种的盐、一种或多于一种的多元醇类有机溶剂和一种或多于一种的表面活性剂；和所述洗脱缓冲液还含有浓度为5mM至20mM的钙离子；和所述洗脱缓冲液还含有10mM至50mM的His-HCL缓冲剂；和所述洗脱缓冲液的pH为4.0至8.0，优选为5.0至6.0，更优选为5.0。
  2. 如权利要求1所述的方法，其特征在于，所述亲和层析步骤在选自下列的亲和介质上进行：MabSelect、MabSelect SuRe、Protein A Diamond和MabSelect SuRe LX；优选地使用MabSelect SuRe LX。
  3. 如权利要求1所述的方法，其特征在于，所述阴离子交换层析步骤在选自下列的强阴离子交换介质上进行：Q HP、Toyopearl GigaCap Q-650、DEAE Beads 6FF、Generik MC-Q、Fractogel EMD TMAE、Q Ceramic HyperD F，优选地使用Q HP。
  4. 如权利要求1所述的方法，其特征在于，所述亲和层析步骤，在融合蛋白洗脱前先使用不含或含有较低浓度的有机溶剂的去污缓冲液淋洗以去除部分污染物；所述去污缓冲液包含20mM His-HCl、0.4-1.5M NaCl、10mM CaCl 2、0.02-0.05％Tween-80、0-10％乙二醇，pH为5.0-7.0；优选为20mM His-HCl、1.5M NaCl、10mM CaCl 2、0.02％Tween-80、3-7％乙二醇，pH为5.0-7.0。
  5. 如权利要求1所述的方法，其特征在于，所述亲和层析步骤中使用的洗脱缓冲液所包含的盐包括氯化钠、氯化铵、氯化钾、硫酸钠、硫酸铵等；优选为氯化物；最优选为盐是氯化钠。
  6. 如权利要求1所述的方法，其特征在于，所述亲和层析步骤中使用的洗脱缓冲液所包含的多元醇类有机溶剂包括乙二醇、1,2-丙二醇和丙三醇，优选为乙二醇；并且所述有机溶 剂的比例为5-40％(w/w)，优选为5-20％。
  7. 如权利要求1所述的方法，其特征在于，所述亲和层析步骤中使用的洗脱缓冲液所包含的钙离子选自可溶性钙盐，包括氯化钙、乙酸钙、乳酸钙、苯甲酸钙；优选为氯化钙。
  8. 如权利要求1所述的方法，其特征在于，所述亲和层析步骤中使用的洗脱缓冲液所包含的表面活性剂为吐温-80(Tween-80)或曲拉通X-100(Triton X-100)，并且所述表面活性剂的浓度为0.01％至0.1％(v/v)，优选为0.02％至0.05％(v/v)。
  9. 如权利要求1所述的方法，其特征在于，所述亲和层析步骤中使用的洗脱缓冲液包含20mM His-HCl、0.5-2.0M NaCl、5-20mM CaCl 2、0.02-0.1％(v/v)Tween-80、2-20％(w/w)乙二醇，和所述洗脱缓冲液的pH为4.5至7.0。
  10. 如权利要求1所述的方法，其特征在于，所述亲和层析具体包括下列步骤：
    (1)用平衡缓冲液(20mM His-HCl、0.2M NaCl、10mM CaCl 2、0.02％Tween-80、pH为6.8-7.2)平衡层析柱3-5个柱体积；
    (2)上样，载量不高于50000IU/ml；
    (3)完成上样后，用上述平衡缓冲液再平衡3-5个柱体积，冲洗未结合的组分；
    (4)使用去污缓冲液(20mM His-HCl、0.5M NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0)淋洗层析柱3-5个柱体积，以去除部分污染物；
    (5)最后用洗脱缓冲液(20mM His-HCl、1.5M NaCl、10mM CaCl 2、0.02％Tween-80、15％乙二醇，pH为5.0)洗脱融合蛋白，收集目标峰。
  11. 如权利要求1所述的方法，其特征在于，所述阴离子交换层析具体包括下列步骤：
    (1)用稀释液(20mM His-HCl、10mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0)将亲和洗脱产物进行稀释；
    (2)再用平衡缓冲液(20mM His-HCl、100mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)平衡层析柱3-5个柱体积；
    (3)上样，上样载量在5000-50000IU/ml；
    (4)上样结束后，用上述平衡缓冲液冲洗层析柱3-5个柱体积；
    (5)用洗脱缓冲液(20mM His-HCl、500mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)冲洗层析柱3-5个柱体积，以去除部分杂蛋白；
    (6)最后用层级梯度的盐离子洗脱缓冲液(20mM His-HCl、1M NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)进行洗脱，条件为洗脱缓冲液从50％至70％，洗脱3-5个柱体积，收集洗脱样品。
  12. 如权利要求1-11任一项所述的方法，其特征在于，所述方法还包括流穿模式的疏水层析步骤，所述疏水层析步骤包括：将所述亲和层析分离产物或所述阴离子交换层析分离产物加载于含四碳或八碳的弱疏水性介质上，然后用平衡缓冲液淋洗层析柱，使上述产物中的杂质及聚合体结合到疏水层析介质上，融合蛋白直接流穿，收集分离产物。
  13. 如权利要求12所述的方法，其特征在于，所述疏水层析步骤在选自下列的弱疏水性介质上进行：Capto Octyl、Octyl Sepharose 4FF、Phenyl Sepharose 6FF；优选地使用Capto Octyl。
  14. 如权利要求12所述的方法，其特征在于，所述疏水层析步骤中使用的平衡缓冲液包含20mM His-HCl、0.1-1.0M NaCl、10mM CaCl 2、0.02％Tween-80、0-10％乙二醇，pH为6.8-7.2。
  15. 如权利要求12所述的方法，其特征在于，所述亲和层析具体包括下列步骤：
    (1)用平衡缓冲液(20mM His-HCl、0.2M NaCl、10mM CaCl 2、0.02％Tween-80、pH为6.8-7.2)平衡层析柱3-5个柱体积；
    (2)上样，载量不高于50000IU/ml；
    (3)完成上样后，用上述平衡缓冲液再平衡3-5个柱体积，冲洗未结合的组分；
    (4)使用去污缓冲液(20mM His-HCl、0.5M NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0)淋洗层析柱3-5个柱体积，以去除部分污染物；
    (5)最后用洗脱缓冲液(20mM His-HCl、1.5M NaCl、10mM CaCl 2、0.02％Tween-80、15％乙二醇，pH为5.0)洗脱融合蛋白，收集目标峰。
  16. 如权利要求12所述的方法，其特征在于，所述疏水层析具体包括下列步骤：
    (1)用稀释液(20mM His-HCl、10mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0)将亲和洗脱产物进行稀释；
    (2)再用平衡缓冲液(20mM His-HCl、0.1-1M NaCl、10mM CaCl 2、0.02％Tween-80、0-8％乙二醇，pH为6.8-7.2)平衡层析柱3-5个柱体积；
    (3)上样，上样载量小于100000IU/ml；
    (4)上样结束后，用上述平衡缓冲液冲洗层析柱3-5个柱体积，收集流穿产物。
  17. 如权利要求12所述的方法，其特征在于，所述阴离子交换层析具体包括下列步骤：
    (1)用稀释液(20mM His-HCl、10mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0)将疏水层析流穿产物进行稀释；
    (2)再用平衡缓冲液(20mM His-HCl、100mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)平衡层析柱3-5个柱体积；
    (3)上样，上样载量在5000-10000IU/ml；
    (4)上样结束后，用上述平衡缓冲液冲洗层析柱3-5个柱体积；
    (5)用洗脱缓冲液(20mM His-HCl、500mM NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)冲洗层析柱3-5个柱体积，以去除部分杂蛋白；
    (6)最后用层级梯度的盐离子洗脱缓冲液(20mM His-HCl、1M NaCl、10mM CaCl 2、0.02％Tween-80，pH为7.0-7.5)进行洗脱，条件为洗脱缓冲液从50％至70％，洗脱3-5个柱体积，收集洗脱样品。
PCT/CN2021/081056 2020-03-31 2021-03-16 高效分离纯化重组人凝血因子VIII Fc融合蛋白的方法 WO2021197051A1 (zh)

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