Eluting buffer solution for purifying recombinant blood coagulation factor VIII and application of eluting buffer solution

Abstract

The invention provides an elution buffer solution for purifying a recombinant blood coagulation factor VIII and application of the elution buffer solution. The elution buffer solution is a buffer system containing copper ions, amino acids and / or pharmaceutically acceptable salts thereof. The concentration of the copper ions is 1-400 [mu] M. The buffer solution is used for separating and purifying rFVIII from a biological sample, the HCD residue is low, the Protein A residue is low, an rFVIII intermediate product or preparation obtained by using the buffer solution is high in specific activity and purity, and an rFVIII separation and purification method developed by using the buffer solution or an rFVIII preparation preparation method developed by using the buffer solution is high in rFVIII activity yield and product yield, beneficial to capacity expansion and suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of protein separation and purification, and in particular to an elution buffer for purifying recombinant coagulation factor VIII and application thereof. Background Art

[0002] Coagulation factor VIII (FVIII), also known as antihemophilic factor, plays a very important role in the endogenous coagulation system and is a cofactor for activating coagulation factor IX. The coagulation factor VIII gene is located at the end of the long arm of chromosome X (Xq28), with a total length of 186kb, containing 25 introns and 26 exons, which encodes a mature protein composed of 2332 amino acids. The FVIII protein can be cleaved by thrombin (IIa) at Arg372, Arg740 and Arg1689 to produce active FVIII (Activated FVIII, FVIIIa), and separated from von Willebrand Factor (vWF). Natural FVIII always combines with vWF to form a complex and exists stably. FVIIIa is a cofactor of activated FIX (FIXa). 2+ In the presence of platelet factor III (PF3), FIXa and FⅧa form an FⅧ complex, which can activate coagulation factor X (FX) in the intrinsic coagulation pathway to produce activated FX (FXa). FXa can be activated in the presence of Ca 2+ In the presence of PF3, it combines with FV to form prothrombin activator, which further activates thrombin to participate in the subsequent coagulation pathway to achieve the purpose of coagulation.

[0003] Hemophilia A is a congenital X-linked bleeding disorder with FVIII activity deficiency. The reduction or loss of FVIII activity leads to dysfunction of the coagulation pathway. Patients have impaired active thrombin generation and prolonged coagulation time. The main clinical manifestations are post-traumatic or spontaneous bleeding in joints, muscles, deep tissues, etc., high rates of joint deformities or disability, and a lifelong tendency to bleed after mild trauma. Severe patients may also experience spontaneous bleeding without obvious trauma. The common treatment for hemophilia A is coagulation factor replacement therapy, which is intravenous injection of coagulation factor VIII or recombinant coagulation factor VIII (rFVIII) derived from human plasma.

[0004] However, during the separation and purification process of recombinant FVIII, there are technical problems such as large FVIII protein loss, low specific activity of intermediates, and high residual host protein. Therefore, in order to meet the needs of optimizing the production process of FVIII products and improve the accessibility of drugs for the prevention / treatment of hemophilia A, it is urgent to develop methods that can improve the purity of recombinant coagulation factor VIII (hereinafter referred to as "rFVIII"). Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides an elution buffer for purifying a recombinant coagulation factor VIII product and its application. The buffer of the present invention is used to separate and purify rFVIII from a biological sample, with low HCD residue and low ProteinA residue. The rFVIII intermediate or preparation obtained by using the buffer has high specific activity and high purity. The rFVIII separation and purification method or rFVIII preparation preparation method developed by using the buffer has high rFVIII activity yield and high product yield, which is conducive to capacity expansion and suitable for large-scale production.

[0006] The present invention is based on the inventor's discovery and understanding of the following facts and problems:

[0007] The prior art discloses that co-expression of rFⅧ protein with truncated vWF-Fc polypeptide (hereinafter referred to as "vWF-Fc") can make rFⅧ highly expressed and stably expressed, and can also improve the recovery rate and quality of rFⅧ in subsequent purification steps. Furthermore, the inventors separated and purified rFⅧ from biological samples containing rFⅧ and vWF-Fc and unexpectedly found that when the elution buffer selected a buffer system containing copper ions of amino acids and / or pharmaceutically acceptable salts thereof, the obtained rFⅧ eluate sample not only had stable activity and high recovery rate, but also significantly reduced HCD residue and ProteinA residue. Further test results show that when the elution buffer contains copper ions, amino acid hydrochloric acid buffer, and amino acids, the above technical effects can be achieved.

[0008] Therefore, in the first aspect of the present invention, the present invention proposes an elution buffer for purifying recombinant coagulation factor VIII. According to an embodiment of the present invention, the elution buffer contains copper ions, amino acids and / or pharmaceutically acceptable salts thereof; the concentration of the copper ions is 1 to 400 μM. The elution buffer according to an embodiment of the present invention is used to separate and purify rFVIII from a biological sample, with low HCD residue and low ProteinA residue. The rFVIII intermediate or preparation obtained by using the elution buffer has high specific activity and high purity. The rFVIII separation and purification method or rFVIII preparation preparation method developed using the elution buffer has high rFVIII activity yield and high product yield, which is conducive to capacity expansion and suitable for large-scale production.

[0009] In a second aspect of the present invention, the present invention provides a buffer combination for preparing recombinant coagulation factor VIII. According to an embodiment of the present invention, the buffer combination comprises: an equilibrium buffer; a washing buffer; and the aforementioned elution buffer.

[0010] It should be noted that the features and advantages described above for the elution buffer are also applicable to this buffer combination and will not be repeated here.

[0011] In the third aspect of the present invention, the present invention provides a method for separating and purifying recombinant coagulation factor VIII. According to an embodiment of the present invention, the method comprises: using the aforementioned elution buffer or the aforementioned elution buffer combination to chromatographically purify a biological sample; the biological sample comprises: recombinant coagulation factor VIII, vWF or Fc fusion protein of truncated vWF.

[0012] It should be noted that the characteristics and advantages described above for the elution buffer and buffer combination are also applicable to this separation and purification method and will not be repeated here.

[0013] In a fourth aspect of the present invention, the present invention provides a method for preparing a recombinant blood coagulation factor VIII preparation. According to an embodiment of the present invention, the method includes: the aforementioned method for separating and purifying recombinant blood coagulation factor VIII.

[0014] It should be noted that the characteristics and advantages described above for the elution buffer, buffer combination, and separation and purification method are also applicable to the preparation method of the preparation and will not be repeated here.

[0015] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. DETAILED DESCRIPTION

[0016] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0017] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0018] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0019] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0020] As used herein, the terms "optional," "optionally," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0021] The present invention provides an elution buffer for purifying recombinant coagulation factor VIII, a buffer combination, a method for separating and purifying recombinant coagulation factor VIII, and a method for preparing a recombinant coagulation factor VIII preparation, which will be described in detail below.

[0022] Elution buffer

[0023] The present invention proposes an elution buffer for purifying recombinant coagulation factor VIII. According to an embodiment of the present invention, the elution buffer contains copper ions, amino acids and / or pharmaceutically acceptable salts thereof; the concentration of the copper ions is 1 to 400 μM. The elution buffer according to an embodiment of the present invention is used to separate and purify rFVIII from a biological sample, with low HCD residue and low ProteinA residue. The rFVIII intermediate or preparation obtained by using the elution buffer has high specific activity and high purity. The rFVIII separation and purification method or rFVIII preparation preparation method developed by using the buffer has high rFVIII activity yield and high product yield, which is conducive to capacity expansion and suitable for large-scale production.

[0024] In this article, the term "intermediate" refers to a pharmaceutical intermediate, and "rFVIII intermediate" refers to a series of components containing the active ingredient of rFVIII from the recombinant expression product of the drug production raw material cell to the final drug during the production process of rFVIII drug. Exemplarily, it includes but is not limited to: rFVIII chromatography eluate.

[0025] In this article, the term "chromatography" refers to a chromatography technique, which is an experimental method for separating and purifying a sample containing a target molecule. For example, the commonly used chromatography techniques for separating and purifying FⅧ include: ion exchange chromatography, affinity chromatography, and hydrophobic chromatography.

[0026] As used herein, the term "chromatography eluate" refers to the liquid obtained after eluting the target molecule from a stationary phase (eg, chromatography medium) using a chromatography elution buffer.

[0027] Herein, the term "chromatography eluate containing FVIII" is equivalent to "FVIII chromatography eluate", which is the liquid obtained after eluting FVIII from the chromatography medium using a chromatography elution buffer. Exemplarily, "rFVIII affinity chromatography eluate" is the liquid obtained after eluting rFVIII from the affinity chromatography medium using an affinity chromatography elution buffer.

[0028] Exemplarily, "rFVIII anion chromatography eluate" is the liquid obtained after eluting rFVIII from anion chromatography medium using anion chromatography elution buffer.

[0029] Exemplarily, "rFVIII hydrophobic chromatography eluate" is the liquid obtained after eluting rFVIII from the hydrophobic chromatography medium using a hydrophobic chromatography elution buffer.

[0030] In this article, the term "Host Cell DNA" refers to the DNA residues of host cells during the production of biological products. These DNAs may originate from the genome or plasmids of host cells. In biological products produced using recombinant DNA technology, such as therapeutic recombinant proteins and monoclonal antibodies, host cell DNA may remain in the final product. Since these residual DNA may contain carcinogenic or immunogenic sequences, they need to be detected, measured and controlled to avoid possible safety risks.

[0031] In this article, the term "Protein A" is a protein widely used in the purification of monoclonal antibodies. It is used because it can specifically bind to the Fc segment of a variety of immunoglobulins. However, during the purification process, protein A may fall off from the solid phase carrier and enter the final antibody product. This detached protein A is called residual protein A (RPA for short). Protein A has high immunogenicity, which may cause an immune response in the human body and lead to adverse immune-related side effects. Therefore, drug regulatory agencies require the detection and control of protein A residues to ensure the safety of drugs.

[0032] In this article, the term "pharmaceutically acceptable" ingredients are suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. Exemplarily, the "pharmaceutically acceptable salt" can include, but is not limited to, hydrochloride, phosphate, citrate, carbonate, lactate, tartrate, maleate, succinate, glutamate, acetate.

[0033] According to an embodiment of the present invention, the concentration of the copper ion is 5-400 μM, preferably 5-250 μM, preferably 75-250 μM, preferably 75-125 μM, and more preferably 100 μM. The inventors have determined the above-mentioned suitable concentration after a large number of experiments. Thus, rFⅧ is eluted using a chromatography elution buffer containing the above-mentioned concentration of copper ions, and the rFⅧ protein has a high recovery rate, high purity, low HCD residue, and low ProteinA residue.

[0034] Exemplarily, the concentration of the copper ions is 1 μM, 5 μM, 15 μM, 25 μM, 35 μM, 45 μM, 55 μM, 65 μM, 75 μM, 85 μM, 95 μM, 105 μM, 115 μM, 125 μM, 135 μM, 145 μM, 155 μM, 165 μM, 175 μM, 185 μM, 195 μM, 205 μM, 215 μM, 225 μM, 235 μM, 245 μM, 255 μM, 265 μM, 275 μM, 300 μM, 325 μM, 350 μM, 375 μM, or 400 μM.

[0035] In some optional embodiments of the present invention, the type of amino acid is selected from at least one of arginine, histidine, lysine, glycine, leucine, glutamic acid, threonine, alanine, valine, tryptophan, methionine, tyrosine, and serine.

[0036] In the present invention, amino acids can also be represented by single-letter or three-letter codes, with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0037] According to an embodiment of the present invention, the pharmaceutically acceptable salt is an amino acid hydrochloride;

[0038] In some specific embodiments of the present invention, the amino acid type in the amino acid hydrochloride is selected from at least one of arginine, histidine, lysine, glycine, threonine, leucine, glutamic acid, alanine, valine, tryptophan, methionine, tyrosine and serine.

[0039] According to an embodiment of the present invention, the concentration of the amino acid is 15-400 mM, preferably 15-300 mM.

[0040] According to an embodiment of the present invention, the elution buffer contains at least two different amino acids and / or amino acid hydrochlorides thereof.

[0041] According to an embodiment of the present invention, the elution buffer contains a first amino acid and a second amino acid, wherein the first amino acid is histidine or a pharmaceutically acceptable salt thereof, and the second amino acid is any one of arginine, threonine, glycine, glutamic acid, methionine or a pharmaceutically acceptable salt thereof.

[0042] According to an embodiment of the present invention, the concentration of the first amino acid is 5-70 mM, preferably 15-50 mM, and more preferably 20-40 mM.

[0043] According to an embodiment of the present invention, the concentration of the second amino acid is 150-400 mM, preferably 200-300 mM, preferably 225 mM-275 mM, and more preferably 250 mM.

[0044] Exemplarily, the concentration of the second amino acid can be selected as: 200mM, 225mM, 235mM, 245mM, 250mM, 265mM or 275mM, 290mM, 305mM.

[0045] According to an embodiment of the present invention, the molar concentration ratio of the first amino acid to the second amino acid is 2: (15-30), preferably 2: 25. Exemplarily, the molar concentration ratio of the first amino acid to the second amino acid may be 2: 15, 2: 20, 2: 25, 2: 30.

[0046] According to an embodiment of the present invention, the pH of the buffer system is 5.5-8.5, preferably pH 6.0-7.0.

[0047] According to an embodiment of the present invention, an acidic solution is used to adjust the pH value of the buffer system.

[0048] Illustratively, the acidic solution includes but is not limited to hydrochloric acid, acetic acid, phosphoric acid, and the like.

[0049] The above examples should not be construed as limiting the present invention.

[0050] Thus, chromatography elution buffers with different amino acid compositions and concentrations are prepared, which is beneficial for the amino acids to fully contact with the biological sample.

[0051] According to an embodiment of the present invention, the buffer further comprises: Ca 2+During the purification and storage of rFⅧ, calcium ions help prevent rFⅧ aggregation and degradation, thereby improving its stability and extending its shelf life.

[0052] According to an embodiment of the present invention, the Ca 2+ The concentration is 100-200 mM, preferably 125-175 mM. Thus, the activity of rFⅧ is further maintained stable and the activity recovery rate is improved.

[0053] For example, Ca 2+ The concentration can be selected as follows: 100mM, 112.5mM, 125mM, 137.5mM, 150mM, 162.5mM, 175mM, 187.5 or 200mM. As known to those skilled in the art, in a specific rFⅧ purification scheme, the technician can determine the optimal calcium ion concentration through experiments to achieve the desired technical effect, such as improving the purity of rFⅧ, reducing HCD and Protein A residues, etc.

[0054] In some optional embodiments of the present invention, the Ca 2+ Any one selected from calcium chloride, calcium nitrate, calcium gluconate, calcium acetate and calcium lactate.

[0055] According to an embodiment of the present invention, the elution buffer further comprises: 100-200 mM Na + ; preferably 125-175 mM Na + Thus, the separation and purification effect of rFⅧ is further improved.

[0056] Exemplarily, the Na + Concentration options: 100mM, 125mM, 150mM, 175mM or 200mM NaCl.

[0057] In some specific embodiments of the present invention, the elution buffer is used for chromatographic purification of recombinant coagulation factor VIII.

[0058] According to an embodiment of the present invention, the chromatography is affinity chromatography. Exemplarily, the affinity chromatography includes but is not limited to Protein A affinity chromatography. The elution buffer according to an embodiment of the present invention is generally applicable to a variety of Protein A affinity chromatography media, and the separation and purification effect of rFⅧ is stable and has good repeatability.

[0059] Herein, the term "Protein A" is a cell wall protein from Staphylococcus aureus that can bind specifically and with high affinity to the Fc portion of a variety of mammalian immunoglobulins, especially IgG. "Protein A affinity chromatography" is a technique for separation and purification using the high affinity interaction between Protein A and specific proteins (especially immunoglobulins), and is a specific type of affinity chromatography. According to an embodiment of the present invention, the filler of the affinity chromatography is selected from, including but not limited to: at least one of EshmuNO A, MabSelectSuRe, MabSelectSuRe LX, AT ProteinADiamond Plus, Nmab Pro, and PraestoJetted A50.

[0060] In some specific embodiments of the present invention, the buffer is used to separate and purify recombinant coagulation factor VIII from a biological sample, wherein the biological sample contains: recombinant coagulation factor VIII, vWF or Fc fusion protein of truncated vWF.

[0061] In this article, the term "truncated protein" refers to a variant form of the protein that is shorter than the normal protein due to a mutation in the gene sequence, such as the premature appearance of a stop codon, a frameshift mutation, or a mutation in a splicing site, which causes the amino acid sequence of the protein to terminate prematurely.

[0062] In this article, "truncated vWF" is equivalent to "vWF truncate" and "truncated recombinant vWF", and is a truncated protein. In this article, truncated vWF should retain partial or complete binding activity with FVIII, be able to form a complex with FVIII, and at least play a role in high and stable expression of rFVIII.

[0063] In some more specific embodiments of the present invention, the amino acid sequence of the Fc fusion protein is shown as SEQ ID NO:2.

[0064] In some more specific embodiments of the present invention, the amino acid sequence of the recombinant coagulation factor VIII is shown as SEQ ID NO:1.

[0065] Buffer Combination

[0066] The present invention provides a chromatography buffer combination, which includes: an equilibrium buffer; a washing buffer; and the aforementioned elution buffer.

[0067] It should be noted that the features and advantages described above for the chromatography elution buffer are also applicable to this chromatography buffer combination and will not be repeated here.

[0068] As used herein, the term "wash buffer" is a buffer used to remove non-specifically adsorbed proteins or other impurities on a chromatography column (such as an affinity chromatography column) while retaining the target protein.

[0069] According to an embodiment of the present invention, the buffer system of the equilibration buffer or the washing buffer is a buffer system containing amino acids and / or pharmaceutically acceptable salts thereof.

[0070] According to an embodiment of the present invention, the type of amino acid is selected from at least one of arginine, histidine, lysine, glycine, threonine, leucine, glutamic acid, alanine, valine, tryptophan, methionine, tyrosine, and serine.

[0071] According to an embodiment of the present invention, the buffer system is histidine or histidine hydrochloride buffer.

[0072] According to an embodiment of the present invention, the concentration of the histidine or histidine hydrochloride is 20-200 mM, preferably 20-100 mM, and more preferably 20-50 mM.

[0073] According to an embodiment of the present invention, the pH of the histidine or histidine hydrochloride buffer system is 5.0 to 8.0, preferably pH 6.0 to 7.0, thereby further improving the stability of the target protein.

[0074] In this article, the term "histidine buffer system" is composed of amino acids (such as glycine, alanine, etc.) directly as buffer components.

[0075] As used herein, the term "histidine hydrochloride buffer system" refers to salts formed by the reaction of amino acids (such as histidine, lysine, etc.) with hydrochloric acid.

[0076] According to an embodiment of the present invention, the equilibration buffer or the washing buffer further comprises: copper ions. The copper ions in the chromatography equilibration buffer or the washing buffer help maintain the stability of rFⅧ activity.

[0077] According to an embodiment of the present invention, the concentration of copper ions is 5 to 400 μM, preferably 75 to 275 μM, preferably 75 to 125 μM, and more preferably 100 μM. Thus, the activity of rFⅧ is further maintained stable, which helps to reduce the residual HCD and ProteinA residues.

[0078] According to an embodiment of the present invention, the copper ions are from one or more of copper nitrate, copper sulfate, copper halide salts and hydrates thereof.

[0079] According to an embodiment of the present invention, the copper halide salt is selected from: one or more of CuCl2, CuBr2, and CuI2, preferably one or more of CuCl2, CuBr2, and CuI2, and more preferably CuCl2.

[0080] According to an embodiment of the present invention, the equilibration buffer or the washing buffer further comprises: Ca 2+ Calcium ions in the chromatography equilibration buffer or elution buffer help maintain the stability of rFⅧ activity.

[0081] According to an embodiment of the present invention, the Ca 2+ The concentration is 2.5-10 mM, preferably 4.5-7.5 mM, and more preferably 5 mM. Thus, the activity of rFVIII is further maintained stable and the activity recovery rate of the rFVIII affinity chromatography eluate is improved.

[0082] For example, the Ca 2+ Concentration options include: 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM, 5.5mM, 6.0mM, 6.5mM, 7.0mM, 7.5mM, 8.0mM, 8.5mM, 9.0mM, 9.5mM or 10.0mM.

[0083] According to an embodiment of the present invention, the Ca 2+ From calcium salts.

[0084] According to an embodiment of the present invention, the equilibration buffer further comprises: 100-200 mM NaCl, preferably 125-175 mM NaCl, thereby avoiding non-specific adsorption of impurity proteins and facilitating elution and purification of rFVIII.

[0085] According to an embodiment of the present invention, the washing buffer further comprises: 250-350 mM NaCl, preferably 275-325 mM NaCl, thereby facilitating the removal of impurity proteins through the washing step and facilitating the elution and purification of rFVIII.

[0086] In some specific embodiments of the present invention, the buffer combination is used to separate and purify recombinant human coagulation factor VIII from a biological sample, wherein the biological sample contains: recombinant coagulation factor VIII, vWF or Fc fusion protein of truncated vWF.

[0087] Separation and purification method

[0088] The present invention provides a method for separating and purifying recombinant human coagulation factor VIII. According to an embodiment of the present invention, the method comprises: using the aforementioned elution buffer or the aforementioned buffer combination to chromatograph a biological sample; wherein the biological sample comprises: recombinant coagulation factor VIII.

[0089] It should be noted that the characteristics and advantages described above for the chromatography elution buffer and the chromatography buffer combination are also applicable to the separation and purification method and will not be repeated here.

[0090] In some specific embodiments of the present invention, the biological sample contains: recombinant coagulation factor VIII, vWF or Fc fusion protein of truncated vWF.

[0091] According to an embodiment of the present invention, the method includes but is not limited to affinity chromatography, anion chromatography, and hydrophobic chromatography.

[0092] Preparation method

[0093] The present invention provides a method for preparing a recombinant human coagulation factor VIII preparation. According to an embodiment of the present invention, the method comprises: the aforementioned method for separating and purifying recombinant human coagulation factor VIII.

[0094] It should be noted that the characteristics and advantages described above for the chromatography elution buffer, chromatography buffer combination, and separation and purification method are also applicable to the preparation method of the preparation and will not be repeated here.

[0095] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0096] Example 1: Expression of recombinant FVIII factor

[0097] In this example, a cell culture expressing human recombinant factor VIII (hereinafter referred to as "rhFVIII") was prepared as follows.

[0098] Referring to the methods of Examples 1 to 3 of Chinese Patent CN111440820B, cell culture supernatant expressing rhFVIII (amino acid sequence as shown in SEQ ID NO: 1) and truncated vWF-Fc polypeptide (amino acid sequence as shown in SEQ ID NO: 2) was obtained.

[0099] The cell culture supernatant was subjected to high-speed centrifugation, deep filtration and S / D virus inactivation to obtain rhFⅧ virus inactivated solution, which was used as the sample for affinity chromatography separation and purification in Example 2. The concentration of S / D reagent was: 0.3-0.4% tributyl phosphate, 1.0-2.0% polysorbate 80, and the inactivation time was: 120-180 min.

[0100] Example 2: Optimization of rhFVIII chromatography elution buffer

[0101] The chromatography column filler can be Eshmuno A / MabSelectSuRe / MabSelectPrismA for affinity chromatography. In this embodiment, the chromatography column filler is MabSelectPrismA, and the column volume is 30.7 mL.

[0102] The equilibration buffer of this embodiment is: 20mM His, 5mM CaCl2, 150mM NaCl, 100μM CuCl2, pH6.5; the elution buffer is 20mM His, 5mM CaCl2, 300mM NaCl, 100μM CuCl2, pH7.0.

[0103] 1. Investigation of amino acid concentration in the chromatography eluent

[0104] In this example, a histidine buffer system (pH 6.5 ± 0.5) containing 150 mM NaCl was selected, and arginine was used as a representative to investigate the effects of different concentrations of amino acids on the affinity chromatography elution of recombinant coagulation factor VIII. The specific scheme is as follows:

[0105] Elution buffer a: 20 mM His, 0.15 M Arg, 150 mM NaCl, 0.15 M CaCl2;

[0106] Elution buffer b: 20 mM His, 0.2 M Arg, 150 mM NaCl, 0.15 M CaCl2;

[0107] Elution buffer c: 20 mM His, 0.3 M Arg, 150 mM NaCl, 0.15 M CaCl2;

[0108] Elution buffer d: 20 mM His, 0.35 M Arg, 150 mM NaCl, 0.15 M CaCl2.

[0109] The pH of the elution buffer was adjusted to 6.5±0.5 with hydrochloric acid.

[0110] After the MabSelectPrismA chromatography column was equilibrated with an equilibration buffer, the rhFⅧ virus inactivated solution of Example 1 was loaded. After loading, the elution buffer was used for elution at a flow rate of 8 mL / min, and then the elution buffers a to d were used for elution at a flow rate of 8 mL / min, and the rhFⅧ eluate was collected. The protein yield, sample purity and impurity content of the rhFⅧ eluate were detected (using the Chinese Pharmacopoeia Phase I method), and the results are shown in Table 1.

[0111] Table 1 Effects of elution buffer containing different concentrations of amino acids on the key quality indicators of rhFⅧ eluate

[0112]

[0113] Note: "Protein recovery rate" refers to the recovery rate of rhFⅧ protein before and after chromatography; "Purity" refers to the purity of rhFⅧ protein in the rhFⅧ eluate; "Specific activity" refers to the specific activity of rhFⅧ in the rhFⅧ eluate; the test results are the average values ​​of the test data of three groups of samples, the same below.

[0114] The above results show that when the ratio of histidine to arginine concentration is within the range of 0.02M:(0.15M-0.3M), the purity, residual impurities and protein recovery rate of the rhFⅧ eluate are not significantly affected, and when the arginine concentration is within the range of 0.2M-0.3M, the residual HCD and residual ProteinA are further reduced. In order to ensure the protein recovery rate and purity, the present invention selects 0.25M Arg for the subsequent experiments.

[0115] 2. Investigation of copper ion addition concentration

[0116] The present application further investigates the concentration of copper ion addition, and the copper ions can be added in the form of compounds such as CuCl2, CuBr2, CuI2, copper nitrate, copper sulfate and their hydrates; in the present embodiment, the copper ions are added in the form of CuCl2.

[0117] The inventors controlled the copper ion concentration in the affinity chromatography elution buffer and adjusted the pH to 6.5±0.5 with hydrochloric acid to investigate the effect of copper ion concentration on the protein yield, sample purity and impurity content of the rhFⅧ affinity chromatography elution buffer. The design scheme and results are shown in Table 2.

[0118] Table 2 Experimental design and results of copper ion concentration optimization

[0119]

[0120] The above results show that during the rhFⅧ chromatography process, as the copper ion concentration in the elution buffer increased, the protein recovery rate and purity increased, and the impurity content gradually decreased; when the concentration was 100μM and 250μM, respectively, there was no significant difference in purity, recovery rate, and impurity content. In subsequent experiments, 100μM copper ions were selected for the next screening test.

[0121] 3. Elution buffer system screening test

[0122] Elution buffer 1: 20 mM His, 0.25 M Arg, 150 mM NaCl, 0.15 M CaCl2, 100 μM CuCl2;

[0123] Elution buffer 2: 20 mM His, 0.25 M Arg, 170 mM NaCl, 0.15 M CaCl2, 100 μM CuCl2;

[0124] Elution buffer 3: 20 mM His, 0.25 M Arg, 140 mM NaCl, 0.15 M CaCl2, 100 μM CuCl2;

[0125] Elution buffer 4: 20 mM His, 0.25 M Threonine, 150 mM NaCl, 0.15 M CaCl2, 100 μM CuCl2;

[0126] Elution buffer 5: 20 mM His, 0.25 M glycine, 150 mM NaCl, 0.15 M CaCl2, 100 μM CuCl2;

[0127] Elution buffer 6: 20 mM His, 0.25 M glutamate, 150 mM NaCl, 0.15 M CaCl2, 100 μM CuCl2;

[0128] Elution buffer 7: 20 mM His, 0.25 M Arg, 150 mM NaCl, 0.15 M CaCl2, 100 μM Cu(NO3)2;

[0129] Elution buffer 8: 20 mM His, 0.25 M Arg, 150 mM NaCl, 0.15 M CaCl2, 100 μM CuSO4.

[0130] The pH of the elution buffer was adjusted to 6.5±0.5 with hydrochloric acid and then affinity chromatography elution was performed. The results are shown in Table 3.

[0131] Table 3 rhFⅧ chromatography eluate protein yield, sample purity and impurity content test results

[0132]

[0133] The above results show that: (1) Although the sodium chloride concentrations in elution buffers 1, 2, and 3 were different, there were no significant differences in protein recovery rate, purity, specific activity, and impurity residues; (2) Compared with elution buffer 1, when arginine in elution buffers 4, 5, and 6 was replaced by threonine, glycine, or glutamic acid, there were no significant differences in protein recovery rate, purity, and HCD residue and protein A residue; (3) Elution buffers 7 and 8 used copper ions in other forms such as nitrate and sulfate, respectively, and there were no significant differences in protein recovery rate, purity, and HCD residue and protein A residue compared with elution buffer 1.

[0134] The above results show that adding amino acid protective agents such as arginine, threonine, glycine, glutamic acid and a specific concentration of copper ions to the affinity chromatography eluent can effectively improve the protein recovery rate, purity and specific activity of the rFⅧ eluent, and significantly reduce the HCD residue and Protein A residue. Thus, the specific activity and purity of rFVIII intermediates or preparations are further improved, and the activity yield and product yield of the rFVIII separation and purification method or preparation method are further improved, which is conducive to capacity expansion and suitable for large-scale production.

[0135] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An elution buffer for purifying a recombinant coagulation factor VIII product, characterized in that: The elution buffer is a buffer system containing copper ions, amino acids and / or pharmaceutically acceptable salts thereof; the concentration of the copper ions is 1 to 400 μM.

2. The elution buffer according to claim 1, characterized in that The concentration of the copper ion is 5 to 400 μM, preferably 5 to 250 μM, preferably 75 to 250 μM, preferably 75 to 125 μM, more preferably 100 μM; Optionally, the copper ions are selected from one or more of copper nitrate, copper sulfate, copper halide salts and hydrates thereof; Optionally, the copper halide salt is selected from: one or more of CuCl2, CuBr2, CuI2, CuCl, CuBr, CuI, preferably one or more of CuCl2, CuBr2, CuI2, more preferably CuCl2.

3. The elution buffer according to claim 1, characterized in that The amino acid is selected from at least one of arginine, histidine, lysine, glycine, threonine, leucine, glutamic acid, alanine, valine, tryptophan, methionine, tyrosine and serine; Optionally, the pharmaceutically acceptable salt is an amino acid hydrochloride; Optionally, the elution buffer contains at least two different amino acids and / or amino acid hydrochlorides thereof; Optionally, the elution buffer contains a first amino acid and a second amino acid, wherein the first amino acid is histidine or a pharmaceutically acceptable salt thereof, and the second amino acid is any one of arginine, threonine, glycine, glutamic acid, methionine or a pharmaceutically acceptable salt thereof; Optionally, the concentration of the first amino acid is 5 to 70 mM, preferably 15 to 50 mM, more preferably 20 to 40 mM; Optionally, the concentration of the second amino acid is 150-400 mM, preferably 200-300 mM, preferably 225 mM-275 mM, more preferably 250 mM; Optionally, the molar concentration ratio of the first amino acid to the second amino acid is 2:(15-30), preferably 2:25; Optionally, the pH of the elution buffer is 5.5 to 8.5, preferably pH 6.0 to 7.

0.

4. The elution buffer according to any one of claims 1 to 3, characterized in that The buffer also includes: Ca 2+ ; Optionally, the Ca 2+ The concentration is 100-200 mM, preferably 125-175 mM; Optionally, the Ca 2+ Any one selected from calcium chloride, calcium nitrate, calcium gluconate, calcium acetate, and calcium lactate; The elution buffer further comprises: 100-200 mM Na + , preferably 125-175 mM Na + .

5. A buffer composition for preparing recombinant coagulation factor VIII, characterized in that: include: Equilibration buffer; Wash buffer; The buffer according to any one of claims 1 to 4.

6. The buffer solution combination according to claim 5, characterized in that: The buffer system of the equilibration buffer or the elution buffer is a buffer system containing amino acids and / or pharmaceutically acceptable salts thereof; Optionally, the type of amino acid is at least one selected from arginine, histidine, lysine, glycine, threonine, leucine, glutamic acid, alanine, valine, tryptophan, methionine, tyrosine, and serine; Optionally, the buffer system is histidine or histidine hydrochloride buffer; Optionally, the concentration of histidine or histidine hydrochloride is 20 to 200 mM, preferably 20 to 100 mM, more preferably 20 to 50 mM; Optionally, the pH of the histidine or histidine hydrochloride buffer system is 5.0 to 8.0, preferably pH 6.0 to 7.0; Optionally, the equilibration buffer or the wash buffer further comprises: copper ions; Optionally, the concentration of the copper ions is 5 to 400 μM, preferably 75 to 275 μM, preferably 75 to 125 μM, more preferably 100 μM; Optionally, the copper ions are selected from one or more of copper nitrate, copper sulfate, copper halide salts and hydrates thereof; Optionally, the copper halide salt is selected from: one or more of CuCl2, CuBr2, CuI2; preferably one or more of CuCl2, CuBr2, CuI2; more preferably CuCl2; Optionally, the equilibration buffer or the wash buffer further comprises: Ca 2+ ; Optionally, the Ca 2+ The concentration is 2.5 to 10 mM, preferably 4.5 to 7.5 mM, more preferably 5 mM; Optionally, the Ca 2+ Derived from a calcium halide salt; further, the calcium halide salt is calcium chloride.

7. The buffer solution combination according to claim 5, characterized in that: The equilibration buffer further comprises: 100-200 mM NaCl, preferably 125-175 mM NaCl; The washing buffer further comprises: 250-350 mM NaCl, preferably 275-325 mM NaCl.

8. A method for separating and purifying recombinant coagulation factor VIII, characterized in that: include: The biological sample is chromatographed and purified using the elution buffer described in any one of claims 1 to 4 or the buffer combination described in any one of claims 5 to 7; wherein the biological sample comprises: recombinant coagulation factor VIII.

9. The method according to claim 8, characterized in that The biological sample comprises: recombinant coagulation factor VIII, vWF or Fc fusion protein of truncated vWF.

10. A method for preparing a recombinant coagulation factor VIII preparation, characterized in that: include: The method for separating and purifying recombinant coagulation factor VIII according to claim 8 or 9.

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