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WO2015067147A1 - 重组蛋白质的纯化方法 - Google Patents

重组蛋白质的纯化方法 Download PDF

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Publication number
WO2015067147A1
WO2015067147A1 PCT/CN2014/090150 CN2014090150W WO2015067147A1 WO 2015067147 A1 WO2015067147 A1 WO 2015067147A1 CN 2014090150 W CN2014090150 W CN 2014090150W WO 2015067147 A1 WO2015067147 A1 WO 2015067147A1
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conductivity
protein
exchange medium
eluent
buffer
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French (fr)
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胡辉
朱云斌
臧美翎
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Shanghai CP Guojian Pharmaceutical Co Ltd
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Shanghai CP Guojian Pharmaceutical Co Ltd
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Priority to EP14860229.5A priority Critical patent/EP3067367B1/en
Priority to JP2016525887A priority patent/JP6456376B2/ja
Priority to US15/034,821 priority patent/US10246484B2/en
Publication of WO2015067147A1 publication Critical patent/WO2015067147A1/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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention belongs to the field of protein purification, and more particularly, to a method for purifying a recombinant protein.
  • Ion exchange chromatography is a chromatographic technique commonly used for protein purification.
  • ion exchange chromatography if the ionic strength of the surrounding buffer is sufficiently low, the charged portion of the solute surface is attracted by the opposite charge bound to the chromatography matrix. Elution is usually achieved by increasing the ionic strength (conductance value) of the buffer and the potential point of the solute competing ion exchange medium.
  • Changing the pH to change the amount of solute charge is another way to achieve solute elution.
  • the change in conductivity or pH can be gradual (gradient elution) or stepwise (step elution). In the past, these changes were gradual, with a one-way increase or decrease in pH or conductivity.
  • Chinese Patent No. 200410068790.3 discloses the use of cation exchange chromatography to remove acidic contaminants, the use of relatively high conductance values to remove acidic contaminants, and then reduce the conductance balance, and then increase Conductance to elute. The pH does not change during use. Purification results showed that the acid mutant decreased by about 50%, and the alkaline mutant was not mentioned.
  • Chinese Patent 200880119331.X discloses the use of cation exchange chromatography to raise the pH to wash, and then lower the pH to increase the conductance to remove CHOP (Chinese hamster ovary protein), shed protein A, DNA, polymer, etc. in the antibody, and does not target antibodies. Acidic, basic related proteins.
  • the invention utilizes changing the pH of the buffer and the concentration of the salt to separate the acidic, basic related proteins and the target protein, and is applied under the conditions of acidic low pH and relatively high salt concentration, in the alkaline high pH and the relatively low salt. Wash and elute under the conditions of the solution.
  • the final acid-related protein removal rate is greater than 85%, even as high as 93%, the alkaline-related protein removal rate is greater than 59%, and the target protein loss rate is less than 26%.
  • the present invention discloses a method for purifying a recombinant protein from a mixture comprising a recombinant protein and a related protein thereof, the method comprising the following steps:
  • the first equilibrium buffer is used to bind the recombinant protein to the ion exchange medium, wherein the first equilibrium solution is at the first conductivity and pH;
  • the above ion exchange medium is a cation exchange medium, and refers to a filler having SO 3 - functional groups bonded to different substrates, which may be, but not limited to, carboxyl-methyl-cellulose, BAKERBOND ABX TM , immobilized on agarose Sulphopropyl (SP) (such as GE's SP-SEPHAROSE FAST FLOW TM or SP-SEPHAROSE HIGH PERFORMANCE TM ), SOURCE-30S, SOURCE immobilized on polystyrene styrene polystyrene/divinyl benzene -15S, AB company Poros HS Poros XS and immobilized on agarose sulfonyl group (e.g., a Pharmacia S-SEPHAROSE FAST FLOW TM) and Bio-rad company NUVIA-S is fixed on the hydrophilic polyacrylamides , UNOsphere-S, etc.
  • SP agarose Sulphopropy
  • the first type of related protein PI (isoelectric point) is lower than the recombinant protein PI, and the recombinant protein PI is lower than the second type of related protein. More specifically, the first type of related protein is an acidic mutant of the recombinant protein, which is defined as The type of material that retained less time than the target protein on CEX-HPLC.
  • the second class of related proteins are basic mutants of recombinant proteins, defined as those that retain a greater time on CEX-HPLC than the target protein.
  • the second equilibrium buffer has lower conductivity than the first equilibrium buffer, but the pH is the same.
  • the first balance solution is a salt-containing buffer, and the commonly used buffer has PB and MES.
  • Acetic acid preferably acetic acid buffer, buffer concentration control range of 10-50mmol / L, preferably 20mmol / L.
  • the pH is controlled at pH 4.0-6.0, and the preferred control range is at pH 4.9-5.1, preferably at pH 5.0.
  • the salt is a salt of sodium chloride, ammonium sulfate, potassium chloride, ammonium chloride or potassium sulfate, and ammonium sulfate is preferred.
  • the salt concentration is controlled in the range of 10-70 mmol/L, preferably 40-60 mmol/L, and the conductance control range is 8-13 ms/cm.
  • the use of 60mmol / L ammonium sulfate is more conducive to the outflow of acid-related proteins;
  • the second balance solution is a salt-free buffer, the commonly used buffers are PB, MES, acetic acid, preferably acetate buffer, buffer
  • the concentration control range is 10-50 mmol/L, preferably 20 mmol/L.
  • the pH is controlled at pH 4.0-6.0, and the preferred control range is at pH 4.9-5.1, preferably at pH 5.0.
  • the conductance is generally 1-2 ms/cm, preferably 1.1 ms/cm.
  • the wash liquor has a lower conductivity than the first equilibration buffer but a higher pH than the first and/or second equilibration buffer.
  • the first eluent has a higher pH than the wash solution, but the conductivity is substantially the same.
  • the second eluent has a higher pH and/or conductivity than the first eluent.
  • the wash solution and the first eluate are achieved by adjusting the mixing ratio of the two different pH salt-containing buffers.
  • the buffer can be selected from phosphate, HEPES, BICINE, etc., preferably phosphate.
  • the buffer concentration control range is 10-50 mmol/L, preferably 10 mmol/L, and the conductance range is generally 1-2 ms/cm, which is generally not controlled.
  • the pH of a buffer A can be between 7.0 and 7.8, preferably at 7.5, and the pH of a buffer B is between 9.3 and 9.4.
  • the salt is a salt of sodium chloride, ammonium sulfate, potassium chloride, ammonium chloride or potassium sulphate; more specifically, the pH of the washing liquid and the first eluate is changed by 25% Na 2 HPO 4 pH 7. 5+75% Na 2 HPO 4 pH 9.3-9.4 was changed to 15% Na 2 HPO 4 pH 7.5 + 85% Na 2 HPO 4 pH 9.3-9.4.
  • the second eluent is a high-salt aqueous solution
  • the salt is a salt of sodium chloride, ammonium sulfate, potassium chloride, ammonium chloride or potassium sulfate, preferably sodium chloride.
  • Various buffers are typically stored at 4-30 degrees Celsius, preferably 4-8 degrees Celsius.
  • the recombinant protein preferably a recombinant antibody (e.g., a recombinant anti-HER2 antibody) is expressed by CHO cells, and the supernatant is collected using a disc centrifugation technique and depth filtration. Then, it is eluted by protein-A affinity chromatography using acidic citric acid to obtain a homogeneous mixture containing the target protein, acidic, and basic related proteins. This mixture is a sample solution of a cationic chromatography (for example, Nuvia-S).
  • a cationic chromatography for example, Nuvia-S.
  • the sample solution is adjusted to pH 4.0-6.0 by alkaline substances such as TRIS-base/sodium hydroxide, and the preferred control range is pH 4.9-5.1, preferably at pH 5.0.
  • alkaline substances such as TRIS-base/sodium hydroxide
  • the preferred control range is pH 4.9-5.1, preferably at pH 5.0.
  • chlorine is added.
  • Salting salt such as sodium/ammonium sulfate to adjust the conductance, using a temperature-compensated conductivity meter, such as METTLER's seven-eas y conductivity meter, using 20 degrees Celsius as the reference temperature, adjusting the conductivity control range at 6.0 -18.0 ms/cm, preferably controlled at 8.0-12.5 ms/cm.
  • the present invention uses two different loading conductances, one being 8.4 ms/cm and the other being 12.0 ms/cm.
  • the use of high conductance loading at the same pH facilitates the outflow of acid-related proteins, generally 10- A 20% ratio of acid peaks flows out and the rate of protein loss is negligible. After the pH and conductance of the sample are adjusted, it is placed at 4 degrees Celsius, which can slow down the further hydrolysis of the protein of interest.
  • the general control column efficiency is above 2000 per meter.
  • the flow rate of the chromatographic process is generally controlled at 5 cm/min.
  • the column loading is controlled at 10-20 mg/ml, preferably at 15 mg/ml.
  • the first equilibrium liquid balance column is used first.
  • the 3CV can be loaded after the balance is completed. After loading, continue to equilibrate at least 1 CV with the first balance solution, and then equilibrate at least 1 CV with the second balance solution.
  • the purpose of this step is to remove the salt from the equilibration solution to prevent the washing step from being affected.
  • the 2 CV was then washed with a 100% A wash to increase the pH.
  • the column was washed with a 60% B-75% B wash to remove acid-related proteins.
  • the washing step can be carried out in a single step or in multiple steps. Generally, at least 75% B single step washing is used, and the multi-step washing is more advantageous for removing the acid-related protein.
  • the pH of the wash is generally between 7.6 and 7.9.
  • the washing process typically lasts 20-30 CV.
  • the washing process is considered to be completed when the washing peak drops to about 50 mAu.
  • the elution was carried out using 85% B first eluent, and the pH of the elution was generally between 8.05 and 8.15.
  • the elution process typically lasts 8-15 CV.
  • the elution process is considered complete when the elution peak drops to around 50 mAu.
  • a second eluent is used to elute the alkaline related protein.
  • the ratio of the acid-related protein in the sample solution is controlled to be 50% or less, and the lower the better.
  • the ratio of the basic peak is controlled to be less than 20%, and the lower the better.
  • the chromatogram can be used to make the main peak of the target protein CEX-HPLC purity From 37% to more than 70%, the purity can be further increased to 75% or even 77% by using the multi-step washing mode or the loading and discharging mode.
  • the yield of the target protein is generally above 74%.
  • a recombinant protein is a protein produced in a host cell which has been transformed or transfected with a nucleic acid encoding the protein or which produces a protein as a result of homologous recombination.
  • Transformation and “transfection” are used interchangeably and refer to the process of introducing a nucleic acid into a cell. After transformation or transfection, the nucleic acid can be integrated into the host cell genome or exist as an extrachromosomal factor.
  • a "host cell” comprises cells in vitro cell culture and cells in a host animal. Methods for recombinant production of polypeptides are described, for example, in U.S. Patent 5,534,615, incorporated herein by reference.
  • the recombinant protein of the present invention mainly refers to an antibody, and particularly refers to all recombinant antibodies that bind to the HER2 antigen, including but not limited to trastuzumab (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992)), pertuzumab (OMNITARGTM) (WO 01/00245), and US Patent No. 64,072,135, US 5,821,337, US 6,639,055, US 6,671,071, US 6,800,738, US 6,054,297, US 5,677,171 All of the antibody proteins mentioned in US Pat. No. 5,770,195, US Pat. No. 5, 720, 954, US Pat.
  • HER2 antigen refers to human HER2 protein, for example, in Semba et al, PNAS (USA) 82: 6497-6501 (1985) and Yamamoto et al, Nature, 319: 230-234 (1986) (GeneBank Accession No. X03363). The human HER2 protein described in .
  • Acid-related protein A variant of a recombinant protein of interest that is more acidic (eg, as determined by cation exchange chromatography) than the recombinant protein of interest.
  • An example of an acid related protein is a deamidation variant.
  • a basic related protein a variant of a recombinant protein of interest that is more basic than the target recombinant protein (for example, as determined by cation exchange chromatography).
  • Examples of basic related proteins are incomplete removal of C-terminal lysine and incomplete cyclization of N-terminal GLN (glutamine).
  • Mixed solution refers to the term "mixture” (preferably an anti-HER2 antibody) comprising a composition of antibodies, meaning the presence of the desired antibody and one or more acidic and basic variants thereof.
  • the acidic variant may comprise a majority of deamidated anti-HER2 antibodies and a small amount of other acidic variants. It has been found that, for example, in preparations derived from recombinantly expressed anti-HER2 antibodies, up to about 50% of the anti-HER2 antibodies are deamidated and about 15% of the antibodies are basic related proteins.
  • Cationic ion exchange medium refers to a negatively charged solid phase which has a free cation and is capable of exchanging cations in an aqueous solution flowing through a solid phase or in a solid phase.
  • the negatively charged ligand attached to the solid phase to form the cation exchange resin may be, for example, a carboxylate or a sulfonate.
  • SO 3 - fillers including but not limited to: carboxy - sulfo cellulose, BAKERBOND ABX TM, immobilized on agarose - methyl Sulphopropyl (SP) (such as GE's SP-SEPHAROSE FAST FLOW TM or SP-SEPHAROSE HIGH PERFORMANCE TM ), SOURCE-30S, SOURCE-15S immobilized on polystyrene styrene polystyrene/divinyl benzene , Porus HS Poros XS from AB and sulfonyl groups immobilized on agarose (such as Pharmacia's S-SEPHAROSE FAST FLOW TM ) and Bio-rad's NUVIA-S and UNOsphere immobilized on hydrophilic polyacrylamides -S and so on.
  • SP agarose - methyl Sulphopropyl
  • SP SP-SEPHAROSE FAST FLOW TM
  • Buffer is a solution that resists changes in pH by the action of its acid-base coupling component. Different buffers that can be used depending on the pH required for the buffer are described in Buffer, Guidelines for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D. Ed. Calbiochem Corporation (1975).
  • the buffer has a pH ranging from about 5 to about 7 (as in Example 1 below). Examples of buffers in which the pH is controlled within this range include: MES, MOPS, MOPSO, phosphate, acetate, citrate, succinate, and ammonium salt buffers, and combinations of these buffers.
  • Sample refers to a buffer used to add a composition containing a protein of interest and one or more related proteins to an ion exchange resin.
  • the loading buffer has a conductivity and/or pH that allows the target protein molecule (and typically one or more contaminants) to bind to the ion exchange resin.
  • Wash Buffer is used to elute one or more related proteins from the ion exchange resin prior to elution of the target protein.
  • the conductivity and/or pH of the wash buffer allows the relevant proteins to be eluted from the ion exchange resin, but the target protein elutes in small amounts.
  • Elution Buffer Used to elute the target protein from the solid phase.
  • the conductivity and/or pH of the elution buffer elutes the target protein from the ion exchange resin.
  • Conductivity refers to the ability of an aqueous solution to conduct electrical current between two electrodes. In solution, current is transported by ions. Therefore, when the amount of ions in the aqueous solution increases, the solution will have a higher conductivity.
  • the conductivity is measured in mmhos/cm (ms/cm) and can be measured with a conductivity meter (sold by Orion et al).
  • the conductivity of the solution can be varied by varying the concentration of ions therein. For example, the concentration of buffer in the solution and/or the concentration of a salt such as NaCl or KCl can be varied to achieve the desired conductivity.
  • Example 1 No flow through the sample, using a single step 75% B wash, single step 85% B elution
  • Sample rhuMAb HER2 antibody and related protein mixture, replaced by r-protein A chromatography into citric acid system, adjusted to pH 5.0 with TRIS-base, adjusted with sodium chloride to adjust the conductance to 8.5ms/cm, and the loading was 15mg/ Ml load.
  • the total sample loading is 750 mg.
  • Peak collection protein amount. The protein concentration of each component was determined by scanning each sample by spectrophotometer (loading, peak 1, peak 2, peak 3, peak 4). The results were used to calculate the amount of product recovered.
  • Peak 1 (washing peak): 0.855L*0.37mg/ml 316.35mg
  • the main peak yield is: 41.48%
  • CEX-HPLC The target peak yield analysis (CEX-HPLC) is shown in Figure 2, and the relevant data is shown in Table 1.
  • the target peak purity can be increased from 37.51% to 69.99% using the sample loading, where:
  • Example 2 did not flow through the sample, using multiple steps of 60% B-70% B-75% B wash, single step 85% B elution
  • Solution B 10 mm Na 2 HPO 4 + phosphoric acid pH: 9.36 Conductivity 1.5 ms/cm
  • the main peak yield is: 37.95%
  • CEX-HPLC The target peak yield analysis (CEX-HPLC) is shown in Figure 4, and the relevant data is shown in Table 2.
  • the target peak purity can be increased from 37.53% to 75.15%, of which:
  • Example 3 flow through, using a single step 75% B wash, single step 85% B elution
  • Solution B 10 mm Na 2 HPO 4 + phosphoric acid pH: 9.36 Conductivity 1.5 ms / cm
  • Peak collection protein amount. The protein concentration of each component was measured by scanning each sample by spectrophotometer (loading, peak 1, peak 2, peak 3, peak 4, peak 5). The results were used to calculate the amount of product recovered.
  • Peak 2 (washing peak): 807ml*0.4388mg/ml 354.19mg
  • Peak 4 (main peak): 418 ml * 0.6507 mg / ml 272 mg
  • CEX-HPLC The target peak yield analysis (CEX-HPLC) is shown in Figure 6, and the relevant data is shown in Table 3.
  • the target peak purity can be increased from 37.59% to 77.34%, where:
  • Example 3 can remove acid peaks and basic peaks more efficiently, but The target protein yield is slightly reduced.
  • Examples 1-3 illustrate the efficiency of combining relatively low pH, high salt loading, relatively high pH, low salt wash, elution in isolation of recombinant proteins and their associated proteins.
  • the rhuMAb HER2 antibody and related protein mixture in the above Examples 1-3 were obtained by recombinantly producing a light chain amino acid sequence comprising SEQ ID NO: 1 described in Chinese Patent No. 200410068790.3 and SEQ ID NO: in CHO cells: Full length human IgG rhuMAb HER2 of the heavy chain amino acid sequence of 2 (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992). After expression of the recombinant antibody by CHO cells, disc centrifugation was used. The supernatant collected by the technique and depth filtration is a mixture of rhuMAb HER2 antibody and related proteins.
  • CEX-HPLC The detection and analysis methods in Examples 1 to 3: CEX-HPLC is detailed as follows:
  • the chromatogram is integrated by the integral software, calculated by the peak area normalization method, and the purity report is issued.

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Abstract

提供了一种从包含重组蛋白和其相关蛋白的混合物中纯化该重组蛋白的方法，包括以下步骤：A、用处于第一电导率和pH的第一种平衡缓冲液使重组蛋白结合到离子交换介质上；B、用处于第二电导率和pH的第二种平衡缓冲液继续平衡已结合蛋白的离子交换介质；C、用处于第三电导率和pH逐步增加的洗涤液洗涤离子交换介质，洗脱下第一类相关蛋白；D、用处于第四电导率和pH的第一种洗脱液洗脱离子交换介质，洗脱下目的重组蛋白；E、用处于第五电导率和pH的第二种洗脱液继续洗脱离子交换介质，洗脱下第二类相关蛋白。该纯化方法不但提高了酸性和碱性相关蛋白去除率，而且降低了目标蛋白损失率。

Description

重组蛋白质的纯化方法 技术领域
本发明属于蛋白纯化领域，更具体地，本发明涉及一种重组蛋白的纯化方法。
背景技术
离子交换层析是一种常用于蛋白质纯化的层析技术。在离子交换层析中，如果周围缓冲液的离子强度足够低，溶质表面的带电部分就被结合在层析基质上的相反电荷所吸引。通常可提高缓冲液的离子强度(电导值)与溶质竞争离子交换介质的带电位点，来实现洗脱。改变pH从而改变溶质带电荷量是实现溶质洗脱的另一种方法。导电率或pH的改变可以是逐渐的(梯度洗脱)或分步的(分步洗脱)。在过去，这些改变是渐进的，pH或导电率单向增大或减小。
目前已经有许多有关利用阳离子层析纯化蛋白的方法，例如中国专利200410068790.3公开了使用阳离子交换层析去除酸性污染物，使用提相对高电导值来去除酸性污染物，然后降低电导平衡，接着再提高电导来洗脱。pH在使用过程中不变。纯化结果为酸性突变体下降了50％左右，碱性突变体没有提及。中国专利200880119331.X公开了使用阳离子交换层析提高pH来洗涤、然后降低pH提高电导来去除抗体中的CHOP(中国仓鼠卵巢蛋白质)、脱落的蛋白A、DNA、聚体等，并没有针对抗体中的酸性、碱性相关蛋白。
上述方法虽然部分达到了纯化蛋白的目的，但存在着酸性和碱性相关蛋白去除率不高，目标蛋白损失率大的问题。
发明内容
本发明利用改变缓冲液的pH和盐的浓度来分离酸性、碱性相关蛋白和目的蛋白，在酸性低pH和相对高盐浓度的溶液条件下上样，在碱性高pH和相对低盐的溶液的条件下洗涤、洗脱。最终酸性相关蛋白的去除率大于85％，甚至高达93％，碱性相关蛋白的去除率大于59％，目标蛋白损失率小于26％。
更具体地，本发明公开了一种从包含重组蛋白和其相关蛋白的混合物中纯化该重组蛋白的方法，该方法包括依次进行的下列步骤：
A、用第一种平衡缓冲液使重组蛋白结合到离子交换介质上，其中第一种平衡液处于第一电导率和pH；
B、用第二种平衡缓冲液继续平衡已结合蛋白的离子交换介质，其中第二种平衡液处于第二电导率和pH；
C、用不同pH的洗涤液洗涤离子交换介质，从而从离子交换介质上洗脱下第一类相关蛋白，其中洗涤液处于第三电导率和pH是逐步增加的；
D、用第一种洗脱液洗脱离子交换介质，从而从离子交换介质上洗脱下目的重组蛋白，其中第一种洗脱液处于第四电导率和pH；
E、用第二种洗脱液继续洗脱离子交换介质，从而从离子交换介质上洗脱下第二类相关蛋白，其中第二种洗脱液处于第五电导率和pH。
其中，上述离子交换介质是阳离子交换介质，指结合在不同基质上的功能基团为SO3 -的填料，可为但不限于羧基-甲基-纤维素、BAKERBOND ABXTM、固定在琼脂糖上的磺酸丙基(sulphopropyl)(SP)(如GE的SP-SEPHAROSE FAST FLOWTM或SP-SEPHAROSE HIGH PERFORMANCETM)、固定在聚苯乙烯二乙烯苯基Polystyrene/divinyl benzene上的SOURCE-30S、SOURCE-15S，AB公司的Poros HS Poros XS和固定在琼脂糖上的磺酰基(如Pharmacia的S-SEPHAROSE FAST FLOWTM)和Bio-rad公司的固定在亲水性聚丙烯酰胺类上的NUVIA-S、UNOsphere-S等。
第一类相关蛋白PI(isoelectric point，等电点)低于重组蛋白PI，重组蛋白PI低于第二类相关蛋白，更具体地，第一类相关蛋白是重组蛋白的酸性突变体，定义为在CEX-HPLC上保留时间小于目标蛋白的那一类物质。第二类相关蛋白是重组蛋白的碱性突变体，定义为在CEX-HPLC上保留时间大于目标蛋白的那一类物质。
上述的第二种平衡缓冲液的电导率比第一种平衡缓冲液低，但pH两者相同，具体地，第一种平衡液为含盐的缓冲液，一般使用的缓冲液有PB、MES、醋酸，最好选择醋酸缓冲液，缓冲液浓度控制范围在10-50mmol/L，最好在20mmol/L。pH控制在pH4.0-6.0，比较好的控制范围在pH4.9-5.1，最好在pH5.0。盐的种类有氯化钠、硫酸铵、氯化钾、氯化铵、硫酸钾等盐，最好选择硫酸铵。盐浓度控制范围在10-70mmol/L，最好在40-60mmol/L，电导控制范围在8-13ms/cm。使用60mmol/L的硫酸铵更有利于酸性相关蛋白的流出；第二种平衡液为不含盐的缓冲液，一般使用的缓冲液有PB、MES、醋酸，最好选择醋酸缓冲液，缓冲液浓度控制范围在10-50mmol/L，最好在20mmol/L。pH控制在pH4.0-6.0，比较好的控制范围在pH4.9-5.1，最好在pH5.0。电导一般在1-2ms/cm，最好在1.1ms/cm。
洗涤液的电导率比第一种平衡缓冲液低，但pH高于第一和/或第二种平衡缓冲液。
第一种洗脱液的pH比洗涤液高，但电导率基本相同。第二种洗脱液的pH和/或电导率比第一种洗脱液高。
洗涤液和第一种洗脱液是通过调整两种不同pH的含盐的缓冲液的混合比例来实现的。缓冲液可以选择磷酸盐、HEPES、BICINE等，最好选择磷酸盐。缓冲液浓度控制范围在10-50mmol/L，最好在10mmol/L，电导范围一般在1-2ms/cm，一般不控制。一种缓冲液A的pH可以在7.0-7.8，最好在7.5，一种缓冲液B的pH在9.3-9.4。盐的种类有氯化钠、硫酸铵、氯化钾、氯化铵、硫 酸钾等盐；更具体地，洗涤液和第一种洗脱液的pH改变是通过25％Na2HPO4 pH7.5+75％Na2HPO4 pH9.3-9.4改变到15％Na2HPO4 pH7.5+85％Na2HPO4pH9.3-9.4来实现的。
第二种洗脱液是高盐的水溶液，盐的种类有氯化钠、硫酸铵、氯化钾、氯化铵、硫酸钾等盐，优选氯化钠。
各种缓冲液存放一般在4-30摄氏度，最好在4-8摄氏度。
重组蛋白，优选重组抗体(例如重组抗HER2抗体)经过CHO细胞表达后，使用碟式离心技术和深层过滤收集上清液。然后通过protein-A亲和层析，使用pH酸性的柠檬酸来洗脱，获得含目标蛋白、酸性、碱性相关蛋白的均一混合液。此混合液即是阳离子层析(例如Nuvia-S)的上样液。
将上样液通过TRIS-base/氢氧化钠等碱性物质调节至pH4.0-6.0，比较好的控制范围在pH4.9-5.1，最好在pH5.0，pH调节完成后，加入氯化钠/硫酸铵等盐析盐来调节电导，使用带温度补偿的电导率仪，例如梅特勒公司的seven-easy电导率仪，使用20摄氏度为参比温度，调节电导控制范围在6.0-18.0ms/cm，最好控制在8.0-12.5ms/cm。
本发明使用两种不同的上样电导，一种为8.4ms/cm，另一种为12.0ms/cm，在相同pH下采用高电导上样有利于酸性相关蛋白的流出，一般可以有10-20％比例的酸性峰流出而目的蛋白流失率可以忽略不计。样品pH和电导调节完毕后放于4摄氏度，这样可以减缓目的蛋白的进一步水解。
层析柱经装填后需要达到使用要求，一般控制柱效在2000每米塔板数以上。层析过程流速一般控制在5cm/min。层析柱载量控制在10-20mg/ml，最好控制在15mg/ml。蛋白定量使用280nm紫外分光光度计，重组抗体，比如抗HER2人源化单克隆抗体消光系数为1.50。
层析控制上先使用第一种平衡液平衡层析柱，一般3CV可以平衡完毕即可上样。上样后继续使用第一种平衡液平衡至少1CV，再用第二种平衡液平衡至少1CV，此步的目的在于去除平衡液中的盐以防止影响洗涤步骤。接着使用100％A的洗涤液洗涤2CV以提高pH。使用60％B-75％B的洗涤液来洗涤层析柱，用以去除酸性相关蛋白。洗涤步骤可以单步洗涤也可以多步洗涤，一般情况至少使用75％B单步洗涤，多步洗涤更有利于去除酸性相关蛋白。洗涤的pH一般在7.6-7.9。洗涤过程通常持续20-30CV。当洗涤峰下降至50mAu左右时即可认为洗涤过程结束。使用85％B第一种洗脱液来进行洗脱，洗脱的pH一般在8.05-8.15之间。洗脱过程一般持续8-15CV。当洗脱峰下降至50mAu左右时即可认为洗脱过程结束。最后使用第二种洗脱液来洗脱碱性相关蛋白。
上样液的酸性相关蛋白的比例控制在50％以下，越低越好。碱性峰比例控制在20％以下，越低越好。通过层析，可以使主峰的目的蛋白CEX-HPLC纯度 由37％提高到70％以上，使用多步洗涤模式或上样流出模式可以使纯度进一步提高到75％甚至77％以上。目的蛋白的得率一般在74％以上。
本发明中，重组蛋白是在宿主细胞中产生的蛋白，该宿主细胞已被编码该蛋白的核酸转化或转染，或作为同源重组的结果产生蛋白。可互换使用“转化”和“转染”，指将核酸引入细胞的过程。在转化或转染后，核酸可以整合到宿主细胞基因组中，或作为染色体外因子存在。“宿主细胞”包含体外细胞培养中的细胞和在宿主动物中的细胞。例如在美国专利5,534,615中描述了重组产生多肽的方法(纳入本文以供参考)。
本发明所述重组蛋白主要指抗体，特别指的是所有结合HER2抗原的重组抗体，包括但不限于曲妥珠单抗(trastuzumab)(Carter et al.，Proc.Natl.Acad.Sci.USA，89：4285-4289(1992))，帕妥珠单抗(pertuzumab)(OMNITARGTM)(WO01/00245)，以及美国专利US 64072135、US 5821337、US 6639055、US 6719971、US 6800738、US 6054297、US 5677171、US 5770195、US 5720954、US 5772997、US 6165464、US 6387371、US 6399063、US 5720937、US 5725856和中国专利CN 01132225.X中提到的所有抗体蛋白。本文所述的“HER2抗原”指人HER2蛋白，例如在Semba等，PNAS(USA)82：6497-6501(1985)和Yamamoto等，自然，319:230-234(1986)(GeneBank登录号X03363)中描述的人HER2蛋白。
酸性相关蛋白：是比目标重组蛋白更加酸性(例如用阳离子交换层析测定)的目标重组蛋白的变体。酸性相关蛋白的例子是脱酰胺变体。
碱性相关蛋白：是比目标重组蛋白更加碱性(例如用阳离子交换层析测定)的目标重组蛋白的变体。碱性相关蛋白的例子是C端赖氨酸不完全去除、N端GLN(谷氨酰胺)不完全环化作用。
混合液：指包含抗体的组合物的术语“混合液”(优选抗HER2抗体)，指存在所需抗体和其一种或多种酸性变体和碱性变体。酸性变体可以包含占多数的脱酰胺抗HER2抗体和少量其它酸性变体。已发现例如在获自重组表达的抗HER2抗体的制备物中，多达约50％的抗HER2抗体是脱酰胺化的，约15％的抗体为碱性相关蛋白。
阳离子离子交换介质：指一种带负电的固相，因其具有自由阳离子，能交换固相上或固相中流过的水溶液中的阳离子。附着在固相上形成阳离子交换树脂的带负电的配体可以是例如羧酸盐或磺酸盐。商业上可购得的阳离子交换树脂是结合在不同基质上的功能基团为SO3 -的填料,包含但不限于：羧基-甲基-纤维素、BAKERBOND ABXTM、固定在琼脂糖上的磺酸丙基(sulphopropyl)(SP)(如GE的SP-SEPHAROSE FAST FLOWTM或SP-SEPHAROSE HIGH PERFORMANCETM)、固定在聚苯乙烯二乙烯苯基Polystyrene/divinyl benzene上的SOURCE-30S、SOURCE-15S，AB公司的Poros HS Poros XS和固定在琼脂糖上的磺酰基(如Pharmacia的S-SEPHAROSE FAST FLOWTM)和Bio-rad公司的 固定在亲水性聚丙烯酰胺类上的NUVIA-S、UNOsphere-S等。
缓冲液：“缓冲液”是通过其酸-碱偶联组分的作用来抵抗pH变化的溶液。在缓冲液，生物系统中缓冲液的制备和使用指南，Gueffroy,D.Ed.CalbiochemCorporation(1975)中描述了取决于缓冲液所需pH而定的可使用的不同缓冲液。在一个实施例中，缓冲液具有的pH范围从大约5到大约7(如下文实施例1)。将pH控制在该范围内的缓冲液的例子包括：MES、MOPS,MOPSO、磷酸盐、乙酸盐、杵檬酸盐、琥珀酸盐和铵盐缓冲液，以及这些缓冲液的组合。
上样液：“上样液”是指用来将含有目标蛋白分子和一种或多种相关蛋白的组合物加到离子交换树脂上的缓冲液。加样缓冲液具有的电导率和/或pH,能使目标蛋白分子(和通常一种或多种污染物)与离子交换树脂结合。
洗涤缓冲液：“洗涤缓冲液”被用来在洗脱目标蛋白前从离子交换树脂上洗脱出一种或多种相关蛋白。洗涤缓冲液的电导率和/或pH能使相关蛋白从离子交换树脂上被洗脱下来，但目标蛋白洗脱量很少。
洗脱缓冲液：用来将目标蛋白从固相上洗脱下来。洗脱缓冲液的电导率和/或pH能使目标蛋白从离子交换树脂上洗脱下来。
电导率指水溶液在两个电极间传导电流的能力。在溶液中，电流是通过离子运输的。因此，当水溶液中离子量增大时，溶液将具有更高的导电率。导电率的度量单位是mmhos/cm(ms/cm)，而且能用导电率计测量(由Orion等出售)。溶液的导电率可通过改变其中的离子浓度而变化。例如，可改变溶液中缓冲剂浓度和/或盐(如NaCl或KC1)的浓度，来达到所需导电率。
附图说明
图1：实施例1得到的终产物的层析图谱；
图2：实施例1各组分的CEX-HPLC图谱；
图3：实施例2得到的终产物的层析图谱；
图4：实施例2各组分的CEX-HPLC图谱；
图5：实施例3得到的终产物的层析图谱；
图6：实施例3各组分的CEX-HPLC图谱；
具体实施方式
以下实施例仅仅对本发明进行进一步说明，不应理解为对本发明的任何限制。
实施例1：上样不流穿，使用单步75％B洗涤、单步85％B洗脱
层析柱：XK16/40，NuviaS，1CV＝50ml，H＝25cm，flow＝10ml/min
层析系统：AKTA-PURIFIER
操作软件：unicorn系统
样品：rhuMAb HER2抗体及相关蛋白混合物，用r-proteinA层析置换到柠檬酸体系中，再用TRIS-base调节pH5.0，加入氯化钠调节电导为8.5ms/cm，载量为15mg/ml载量。总上样量为750mg。
溶液：
平衡液1：20mmHAC-NaOH+40mm硫酸铵   pH  5.0    电导8.4ms/cm
平衡液2：20mmHAC-NaOH              pH  5.0     电导1.1ms/cm
A溶液：  10mmNa2HPO4+磷酸           pH：7.52    电导1.5ms/cm
B溶液：  10mmNa2HPO4+磷酸           pH：9.36    电导1.5ms/cm
洗涤液1:  100％A
洗涤液2： 25％A  +  75％B
洗脱液1： 15％A  +  85％B
洗脱液2： 300mmol/L NaCl         电导26ms/cm
操作流程：
平衡1(3CV)-上样-平衡1(1CV)-平衡2(2CV)-洗涤液1(2CV)-洗涤液2(20CV)-洗脱液1(12CV))-洗脱液2(2CV)-2N氯化钠(2CV)-纯化水(1CV)-0.1N氢氧化钠(3CV) CV＝柱床
收集峰：蛋白量.通过分光光度计扫描各样品测定各组份的蛋白浓度，(上样，峰1，峰2，峰3，峰4)。将结果用来计算产物回收量。
峰1(洗涤峰)：0.855L*0.37mg/ml＝316.35mg
峰2(主峰前)：27ml*0.31mg/ml＝8.37mg
峰3(主峰)：520ml*0.6mg/ml＝312mg
峰4(300mm氯化钠洗脱)：50ml*1.96mg/ml＝98mg
总回收率为：734.72/752*100％＝97.7％
主峰得率为：41.48％
目标峰得率分析(CEX-HPLC)见图2，相关数据见表1。
表1：
Figure PCTCN2014090150-appb-000001
层析图谱见图1。
结果如下：
使用上样不流穿，使用单步75％B洗涤、单步85％B洗脱的方法，可以使目标峰纯度由37.51％提高到69.99％，其中：
酸性峰去除率为(1-53.15/359.25)*100％＝85.22％
碱性峰去除率为(1-40.12/109.43)*100％＝63.4％
目标峰损失率为(1-218.37/281.33)*100％＝22.38％。
实施例2上样不流穿，使用多步60％B-70％B-75％B洗涤、单步85％B洗脱
层析柱：XK16/40  NuviaS  1CV＝50ml  H＝25cm  flow＝10ml/min
层析系统：AKTA-PURIFIER
操作软件：unicorn系统
样品：rhuMAb HER2抗体及相关蛋白混合物，用r-proteinA层析置换到柠檬酸体系中，再用TRIS-base调节pH 5.0，加入氯化钠调节电导为8ms/cm，载量为15mg/ml载量。总上样量为745mg。
溶液：
平衡液1：20mmHAC-NaOH+40mm硫酸铵     pH  5.0     电导8.4ms/cm
平衡液2：20mmHAC-NaOH                pH 5.03    电导1.1ms/cm
A溶液：10mmNa2HPO4+磷酸              pH：7.52     电导1.5ms/cm
B溶液：10mmNa2HPO4+磷酸              pH：9.36     电导1.5ms/cm
洗涤液1:100％A
洗涤液2：40％A+60％B
洗涤液3：30％A+70％B
洗涤液4：25％A+75％B
洗脱液1：15％A+85％B
洗脱液2：300mmol/L NaCl    电导26ms/cm
操作流程：
平衡1(3CV)-上样-平衡1(1CV)-平衡2(2CV)-洗涤液1(2CV)-洗涤液2-4等梯度(60％B(5CV)-70％B(7CV)-75％B(12CV))-洗脱液1 85％B(9CV))-洗脱液2(2CV)-2N氯化钠(2CV)-纯化水(1CV)-0.1N氢氧化钠(3CV)
收集峰如下：
峰1(洗涤峰，含75％B洗涤前所有峰)：1.1L*0.31mg/ml＝341mg
峰2(主峰前，85％B洗脱至150mau)：28ml*0.3mg/ml＝8.4mg
峰3(主峰，85％B洗脱从150mau至50mau)：367ml*0.77mg/ml＝282.59mg
峰4(300mm氯化钠洗脱)：53ml*1.48mg/ml＝78.44mg
总回收率为：710.43/7458*100％＝95.35％
主峰得率为：37.95％
目标峰得率分析(CEX-HPLC)见图4，相关数据见表2。
表2：
Figure PCTCN2014090150-appb-000002
层析图谱见图3；
结果如下：
使用上样不流穿，使用多步60％B-70％B-75％B洗涤、单步85％B洗脱的方法，可以使目标峰纯度由37.53％提高到75.15％，其中：
酸性峰去除率为(1-26.76/358.13)*100％＝92.53％
碱性峰去除率为(1-43.46/107.77)*100％＝59.67％。
目标峰损失率为(1-212.37/279.90)*100％＝24.13％。
实施例3上样流穿，使用单步75％B洗涤、单步85％B洗脱
层析柱：XK16/40  NuviaS  1CV＝50ml  H＝25cm  flow＝10ml/min
层析系统：AKTA-PURIFIER
操作软件：unicorn系统
样品：rhuMAb HER2抗体及相关蛋白混合物，用r-proteinA层析置换到柠檬酸体系中，再用TRIS-base调节pH5.0，加入氯化钠调节电导为12ms/cm/cm，载量为15mg/ml载量。总上样量为753.28mg。
溶液：
平衡液1：20mmHAC-NaOH+60mm硫酸铵     pH  5.0     电导12ms/cm
平衡液2：20mmHAC-NaOH               pH  5.0      电导0.9ms/cm
溶液A：10mmNa2HPO4+磷酸             pH：7.50     电导1.5ms/cm
溶液B：10mmNa2HPO4+磷酸              pH：9.36     电导1.5ms/cm
洗涤液1:100％A
洗涤液2：25％A+75％B
洗脱液1：15％A+85％B
洗脱液2：300mmol/L NaCl  电导26ms/cm
操作流程：
平衡1(3CV)-上样-平衡1(1CV)-平衡2(2CV)-洗涤液1(2CV)-洗涤液2(30CV)-洗脱液1(11CV)-洗脱液2(2CV)-2N氯化钠(2CV)-纯化水(1CV)-0.1N氢氧化钠(3CV)
收集峰：蛋白量.通过分光光度计扫描各样品测定各组份的蛋白浓度，(上样，峰1，峰2，峰3，峰4，峰5)。将结果用来计算产物回收量。
峰1(流出峰)：150ml*0.1004mg/ml＝15.06mg
峰2(洗涤峰)：807ml*0.4388mg/ml＝354.19mg
峰3(主峰前)：60ml*0.13mg/ml＝8mg
峰4(主峰)：418ml*0.6507mg/ml＝272mg
峰5(300mm氯化钠洗脱)：51ml*1.96mg/ml＝100mg
总回收率为：749.25/753.28*100％＝99.46％
主峰得率为：36.11％
目标峰得率分析(CEX-HPLC)见图6，相关数据见表3。
表3：
Figure PCTCN2014090150-appb-000003
层析图谱见图5。
结果表明：
使用上样流穿，使用单步75％B洗涤、单步85％B洗脱的方法，可以使目标峰纯度由37.59％提高到77.34％，其中：
酸性峰去除率为(1-22.28/358.56)*100％＝93.78％
碱性峰去除率(1-39.33/111.56)*100％＝64.75％
目标峰得率在(1-210.36/281.36)*100％＝25.23％
相对于实施例1和2，实施例3可以更加高效的去除酸性峰和碱性峰，但会 使目标蛋白得率轻微下降。实施例1-3说明了结合相对低pH高盐上样，相对高pH低盐洗涤、洗脱在分离重组蛋白和其相关蛋白的高效性。
上述实施例1-3中的rhuMAb HER2抗体及相关蛋白混合物是这样获得的：在CHO细胞中重组产生含有中国专利号200410068790.3中所述的SEQ ID NO:1的轻链氨基酸序列和SEQ ID NO:2的重链氨基酸序列的全长人IgG rhuMAb HER2(Carter et al.，Proc.Natl.Acad.Sci.USA，89：4285-4289(1992)。重组抗体经过CHO细胞表达后，使用碟式离心技术和深层过滤收集的上清液即为rhuMAb HER2抗体及相关蛋白的混合物。
申请人还根据美国专利US 64072135、US 5821337和中国专利CN 01132225.X中所提到的rhuMAb HER2抗体序列按照上述方法制备样品，并采用实施例1-3中相同的方法进行检测和验证，最终得到的结果和结论基本是相同的。
实施例1～3中检测分析方法：CEX-HPLC详述如下：
1、试剂和仪器
所有化学试剂纯度级别至少为分析纯。
1.1羧肽酶B：浓度5mg/mL
1.21mol/L Tris.HCl溶液:pH7.4～7.6
1.320mmol/L磷酸盐缓冲液：pH6.4～6.6
1.420mmol/L磷酸盐缓冲液+200mmol/L NaCI：pH6.4-66
1.5高效液相色谱仪:Waters Alliance 2998+2695.Ultimate 3000B系列或其它相同类型液相色谱仪
1.6色谱柱:DionexPropac WCX-10(4X250mm)
2试验步骤
2.1供试品处理
2.1.1羧肽酶B酶切处理：对供试品进行酶切处理。反应体积100～500uL,溶液含anti-HER2rhMAb样品5mg/mL，10％(V/V)1mol/L Tris-HCl缓冲液pH7.5和2％(V/V)羧肽酶B(5mg/mL),37℃水浴反应3小时。
2.1.2进样前稀释：上步反应溶液均采用20mmol/L磷酸盐缓冲液(pH6.4～6.6)稀释至1.0mg/mL,12000r/min离心10min,取上清进样。
2.2色谱条件
2.2.1柱温:40±2.0℃
2.2.2样品温度:5±3.0℃
2.2.3检测波长：214nm
2.2.4进样体积：10uL
2.2.5流速：1.0mL/min
2.2.6流动相：A：20mmol/L磷酸盐缓冲液(pH6.4～6.6)；B：20mmol/L磷酸盐缓冲液(pH6.4-66)+200mmol/L NaCI.配制后经0.22um滤膜抽滤、超声、脱气，于2-8摄氏度保存。
2.2.7流动相梯度表
Figure PCTCN2014090150-appb-000004
3结果分析
色谱图按积分软件积分处理，按峰面积归一化法计算，出具纯度报告。

Claims (24)

  1. 一种从包含重组蛋白和其相关蛋白的混合物中纯化该重组蛋白的方法，其特征在于，该方法包括依次进行的下列步骤：
    A.用第一种平衡缓冲液使重组蛋白结合到离子交换介质上，其中第一种平衡液处于第一电导率和pH，
    B.用第二种平衡缓冲液继续平衡已结合蛋白的离子交换介质，其中第二种平衡液处于第二电导率和pH，
    C.用不同pH的洗涤液洗涤离子交换介质，从而从离子交换介质上洗脱下第一类相关蛋白，其中洗涤液处于第三电导率和pH是逐步增加的，
    D.用第一种洗脱液洗脱离子交换介质，从而从离子交换介质上洗脱下目的重组蛋白，其中第一种洗脱液处于第四电导率和pH，
    E.用第二种洗脱液继续洗脱离子交换介质，从而从离子交换介质上洗脱下第二类相关蛋白，其中第二种洗脱液处于第五电导率和pH。
  2. 如权利要求1所述的方法，其特征在于，所述离子交换介质是阳离子交换介质。
  3. 如权利要求2所述的方法，其特征在于，所述阳离子交换介质是结合在不同基质上的功能基团为SO3 -的填料。
  4. 如权利要求1所述的方法，其特征在于，所述第二种平衡缓冲液的电导率比第一种平衡缓冲液低，但pH两者相同。
  5. 如权利要求1所述的方法，其特征在于，所述洗涤液的电导率比第一种平衡缓冲液低，但pH高于第一和/或第二种平衡缓冲液。
  6. 如权利要求1所述的方法，其特征在于，所述第一种洗脱液的pH比洗涤液高，但电导率基本相同。
  7. 如权利要求1所述的方法，其特征在于，所述第二种洗脱液的pH和/或电导率比第一种洗脱液高。
  8. 如权利要求1所述的方法，其特征在于，所述第一类相关蛋白和目的重组蛋白的洗脱是通过改变洗涤液和第一种洗脱液的pH来实现的。
  9. 如权利要求8所述的方法，其特征在于，所述洗涤液和第一种洗脱液的pH是通过调整两种不同pH的缓冲盐的混合比例来实现改变的。
  10. 如权利要求9所述的方法，其特征在于，所述洗涤液和第一种洗脱液的pH是通过 25％Na2HPO4 pH7.5+75％Na2HPO4 pH9.3-9.4改变到15％Na2HPO4pH7.5+85％Na2HPO4 pH9.3-9.4来实现改变的。
  11. 如权利要求1所述的方法，其特征在于，在步骤A-D中，pH是逐步上升的。
  12. 如权利要求1所述的方法，其特征在于，所述重组蛋白和相其关蛋白是具有不同的PI，第一类相关蛋白的PI低于重组蛋白PI，重组蛋白的PI低于 第二类相关蛋白。
  13. 如权利要求1所述的方法，其特征在于，所述第一类相关蛋白是重组蛋白的酸性突变体，为在CEX-HPLC上保留时间小于目标蛋白的那一类物质，所述第二类相关蛋白是重组蛋白的碱性突变体，为在CEX-HPLC上保留时间大于目标蛋白的那一类物质。
  14. 如权利要求1-13任一项所述的方法，其特征在于，所述的重组蛋白是抗体。
  15. 如权利要求14所述的方法，其特征在于，所述的抗体选自曲妥珠单抗、帕妥珠单抗和其他结合HER2抗原的重组抗体。。
  16. 如权利要求1-15任一项所述的方法，其特征在于，该方法还包括将包含所述重组蛋白和其相关蛋白的混合物在离子交换层析之前、之中或之后经过一次或多次进一步的纯化步骤，来获得该重组蛋白的均一制备物。
  17. 一种从包含重组抗HER2抗体和其相关蛋白的混合物中纯化该重组抗体的方法，其特征在于，该方法包括依次进行的下列步骤：
    a)用第一种平衡缓冲液使重组蛋白结合到阳离子交换介质上，其中第一种平衡液处于第一电导率和pH，
    b)用第二种平衡缓冲液继续平衡已结合蛋白的阳离子交换介质，其中第二种平衡液处于第二电导率和pH，
    c)用不同pH的洗涤液洗涤离子交换介质，从而从阳离子交换介质上洗脱下第一类相关蛋白，其中洗涤液处于第三电导率和pH是逐步增加的，
    d)用第一种洗脱液洗脱离子交换介质，从而从阳离子交换介质上洗脱下目的重组蛋白，其中第一种洗脱液处于第四电导率和pH，
    e)用第二种洗脱液继续洗脱离子交换介质，从而从阳离子交换介质上洗脱下第二类相关蛋白，其中第二种洗脱液处于第五电导率和pH。
  18. 如权利要求17所述的方法，其特征在于，阳离子交换介质是结合在不同基质上的功能基团为SO3 -的填料。
  19. 如权利要求18所述的方法，其特征在于，所述阳离子交换介质选自羧基-甲基-纤维素、BAKERBOND ABXTM、固定在琼脂糖上的磺酸丙基、固定在聚苯乙烯二乙烯苯基Polystyrene/divinyl benzene上的磺酸丙基、固定在琼脂糖上的磺酰基和固定在亲水性聚丙烯酰胺类上的磺酸丙基中的至少一种。
  20. 如权利要求17-19任一项所述的方法，其特征在于，所述第一种平衡缓冲液为含盐的缓冲液，缓冲液为10-50mmol/L醋酸缓冲液，盐是40-60mmol/L氯化钠或硫酸铵，pH 4-6，电导8-13ms/cm，所述第二种平衡缓冲液为不含盐的缓冲液，缓冲液为10-50mmol/L醋酸缓冲液，pH与第一种平衡缓冲液相同，电导1-2ms/cm。
  21. 如权利要求17-19任一项所述的方法，其特征在于，所述洗涤液的电 导率比第一种平衡缓冲液低，但pH高于第一和/或第二种平衡缓冲液，所述第一种洗脱液的pH比洗涤液高，但电导率基本相同，所述第二种洗脱液的pH和/或电导率比第一种洗脱液高。
  22. 如权利要求17-19任一项所述的方法，其特征在于，所述洗涤液和第一种洗脱液的pH是通过调整两种不同pH的缓冲盐的混合比例来实现改变的，通过从 25％Na2HPO4 pH7.5+75％Na2HPO4 pH9.3-9.4改变到15％Na2HPO4pH7.5+85％Na2HPO4 pH9.3-9.4来实现改变的。
  23. 如权利要求17所述的方法，其特征在于，所述重组抗HER2抗体选自曲妥珠单抗、帕妥珠单抗和其他结合HER2抗原的重组抗体。
  24. 如权利要求17-23任一项所述的方法，其特征在于，该方法还包括将包含所述重组抗HER2抗体和其相关蛋白的混合物在阳离子交换层析之前、之中或之后经过一次或多次进一步的纯化步骤，来获得该重组抗HER2抗体的均一制备物。
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Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2016355739A1 (en) * 2015-11-18 2018-07-05 Merck Patent Gmbh Improved protein separation in ion exchange chromatography
RU2018121657A (ru) * 2015-11-18 2019-12-19 Мерк Патент Гмбх ПРОТИВОПОЛОЖНЫЕ ГРАДИЕНТЫ pH-СОЛЬ ДЛЯ УЛУЧШЕННОГО РАЗДЕЛЕНИЯ БЕЛКОВ
CN108101987A (zh) * 2017-11-17 2018-06-01 安徽未名生物医药有限公司 一种重组抗TNF-α全人源单克隆抗体的纯化方法
CN113166200B (zh) * 2018-12-21 2024-03-01 上海药明生物技术有限公司 一种提高蛋白a层析法去除聚集体的方法
CA3160208A1 (en) * 2019-11-07 2021-05-14 Amgen Inc. High salt washes during cation exchange chromatography to remove product-realated impurities
BR112022008649A2 (pt) * 2019-11-07 2022-07-19 Amgen Inc Condicionamento de carga com alto teor de sal durante cromatografia de troca catiônica para remover impurezas relacionadas ao produto
CN111205362A (zh) * 2020-03-19 2020-05-29 贾芳苗 牛血清白蛋白及其制备方法
EP4314025A2 (en) 2021-04-01 2024-02-07 Vestaron Corporation Av3 mutant polypeptides for pest control
WO2023122805A1 (en) 2021-12-20 2023-06-29 Vestaron Corporation Sorbitol driven selection pressure method
US20250212890A1 (en) 2022-03-30 2025-07-03 Vestaron Corporation COMBINATIONS OF AV3 MUTANT POLYPEPTIDES AND Bt TOXINS FOR PEST CONTROL
TW202413392A (zh) 2022-05-18 2024-04-01 美商韋斯塔隆公司 殺蟲ａｃｔｘ肽變異體
WO2023245100A1 (en) 2022-06-17 2023-12-21 Vestaron Corporation Antimicrobial ncr13 variant peptides
CN115073549B (zh) * 2022-07-07 2023-10-20 澳斯康生物(南通)股份有限公司 Hek293细胞裂解液的纯化方法
WO2024026406A2 (en) 2022-07-29 2024-02-01 Vestaron Corporation Next Generation ACTX Peptides

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5534615A (en) 1994-04-25 1996-07-09 Genentech, Inc. Cardiac hypertrophy factor and uses therefor
US5677171A (en) 1988-01-12 1997-10-14 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US5720937A (en) 1988-01-12 1998-02-24 Genentech, Inc. In vivo tumor detection assay
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US6054297A (en) 1991-06-14 2000-04-25 Genentech, Inc. Humanized antibodies and methods for making them
WO2001000245A2 (en) 1999-06-25 2001-01-04 Genentech, Inc. HUMANIZED ANTI-ErbB2 ANTIBODIES AND TREATMENT WITH ANTI-ErbB2 ANTIBODIES
CN1299370A (zh) * 1998-05-06 2001-06-13 基因技术股份有限公司 用离子交换层析纯化蛋白质
US6800738B1 (en) 1991-06-14 2004-10-05 Genentech, Inc. Method for making humanized antibodies
CN102887955A (zh) * 2006-04-05 2013-01-23 艾博特生物技术有限公司 抗体纯化

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1308456B1 (en) * 1998-05-06 2007-08-22 Genentech, Inc. Antibody purification by ion exchange chromatography
JP5303092B2 (ja) * 2002-09-11 2013-10-02 ジェネンテック， インコーポレイテッド タンパク質精製
US7563600B2 (en) * 2002-09-12 2009-07-21 Combimatrix Corporation Microarray synthesis and assembly of gene-length polynucleotides
US20090148435A1 (en) * 2007-10-30 2009-06-11 Genentech, Inc. Antibody purification by cation exchange chromatography
US20120177640A1 (en) * 2009-07-24 2012-07-12 Josef Burg Optimizing the production of antibodies
EP2480561B1 (en) * 2009-09-23 2016-07-13 E. R. Squibb & Sons, L.L.C. Cation exchange chromatography
WO2013054250A1 (en) * 2011-10-10 2013-04-18 Dr Reddy's Laboratories Limited Purification method
WO2013075740A1 (en) * 2011-11-23 2013-05-30 Sanofi Antibody purification method
BR112014011900A2 (pt) * 2011-12-15 2017-05-16 Hanwha Chemical Corp um método de purificação de anticorpo
TW201444867A (zh) * 2013-03-08 2014-12-01 Lilly Co Eli 抗ｔｎｆ－抗ｉｌ－１７雙特異性抗體

Patent Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6387371B1 (en) 1988-01-12 2002-05-14 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US6399063B1 (en) 1988-01-12 2002-06-04 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US5720954A (en) 1988-01-12 1998-02-24 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US6165464A (en) 1988-01-12 2000-12-26 Genetech, Inc. Monoclonal antibodies directed to the HER2 receptor
US5725856A (en) 1988-01-12 1998-03-10 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US5770195A (en) 1988-01-12 1998-06-23 Genentech, Inc. Monoclonal antibodies directed to the her2 receptor
US5772997A (en) 1988-01-12 1998-06-30 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US5720937A (en) 1988-01-12 1998-02-24 Genentech, Inc. In vivo tumor detection assay
US5677171A (en) 1988-01-12 1997-10-14 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US6054297A (en) 1991-06-14 2000-04-25 Genentech, Inc. Humanized antibodies and methods for making them
US6407213B1 (en) 1991-06-14 2002-06-18 Genentech, Inc. Method for making humanized antibodies
US6639055B1 (en) 1991-06-14 2003-10-28 Genentech, Inc. Method for making humanized antibodies
US6719971B1 (en) 1991-06-14 2004-04-13 Genentech, Inc. Method for making humanized antibodies
US6800738B1 (en) 1991-06-14 2004-10-05 Genentech, Inc. Method for making humanized antibodies
US5534615A (en) 1994-04-25 1996-07-09 Genentech, Inc. Cardiac hypertrophy factor and uses therefor
CN1299370A (zh) * 1998-05-06 2001-06-13 基因技术股份有限公司 用离子交换层析纯化蛋白质
WO2001000245A2 (en) 1999-06-25 2001-01-04 Genentech, Inc. HUMANIZED ANTI-ErbB2 ANTIBODIES AND TREATMENT WITH ANTI-ErbB2 ANTIBODIES
CN102887955A (zh) * 2006-04-05 2013-01-23 艾博特生物技术有限公司 抗体纯化

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BUFFERS: "A Guide for the Preparation and Use of Buffers in Biological Systems", 1975, CALBIOCHEM CORPORATION
CARTER ET AL., PROC.NATL.ACAD.SCI.USA, vol. 89, 1992, pages 4285 - 4289
See also references of EP3067367A4
SEMBA ET AL., PNAS (USA, vol. 82, 1985, pages 6497 - 6501
YAMAMOTO ET AL., NATURE, vol. 319, 1986, pages 230 - 234

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CN104628846A (zh) 2015-05-20
JP2016538264A (ja) 2016-12-08
JP6456376B2 (ja) 2019-01-23
US20160289264A1 (en) 2016-10-06
EP3067367A4 (en) 2017-09-13
US10246484B2 (en) 2019-04-02
CN104628846B (zh) 2019-12-06
EP3067367A1 (en) 2016-09-14
EP3067367B1 (en) 2022-05-04

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