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WO2014081225A1 - Procédé de préparation d'un nouveau monomère de l'hormone de croissance humaine à action prolongée - Google Patents

Procédé de préparation d'un nouveau monomère de l'hormone de croissance humaine à action prolongée Download PDF

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Publication number
WO2014081225A1
WO2014081225A1 PCT/KR2013/010630 KR2013010630W WO2014081225A1 WO 2014081225 A1 WO2014081225 A1 WO 2014081225A1 KR 2013010630 W KR2013010630 W KR 2013010630W WO 2014081225 A1 WO2014081225 A1 WO 2014081225A1
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Prior art keywords
growth hormone
human growth
nexp
hgh
protein
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Korean (ko)
Inventor
안지원
강길부
전창봉
이동억
유정민
최은영
김경화
김은영
조성유
박영준
김기완
이윤정
박순재
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CJ CheilJedang Corp
Alteogen Inc
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CJ CheilJedang Corp
Alteogen Inc
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Priority to CN201380060578.XA priority Critical patent/CN104812776A/zh
Publication of WO2014081225A1 publication Critical patent/WO2014081225A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin

Definitions

  • the present invention relates to a method for producing a high purity monomer of NexP-hGH protein, a sustained human growth hormone using anion exchange chromatography, and a monomer of the NexP-hGH protein prepared by the above method.
  • Human growth hormone is a protein hormone with a molecular weight of approximately 21,500 Da consisting of 191 amino acids, and is responsible for promoting growth by stimulating cell differentiation of cartilage growth plates in the body. If there is a deficiency in the synthesis and secretion of human growth hormone in the body, it may cause hypotensive, increased risk of cardiovascular disease, muscle and bone density decrease.
  • the present inventors also endeavored to develop a sustained human growth hormone with increased half-life in the body by maintaining the persistence in the body, and as a result developed a sustained human growth hormone NexP-hGH.
  • the preparation method and protein sequence thereof are disclosed in Korean Patent Registration No. 10-1183262.
  • the long-acting human growth hormone NexP-hGH is an N-terminus or C-terminus of human growth hormone that replaces NexP with specific amino acids to eliminate the intrinsic activity of the protein alpha-1 antitrypsin (A1AT) and increase its half-life. It is a protein fused by genetic recombination.
  • NexP-hGH has the advantage of improved half-life in the body compared to first-generation human growth hormone, and it is made to be glycosylated when expressed in animal cells, CHO cells, and it is different from most first-generation human growth hormone drugs manufactured in Escherichia coli.
  • hGH fused with NexP still retains the structural and chemical properties of hGH itself.
  • hGH is known as a well-formed protein, and hGH in polymer form is known to not only induce antigenicity in the body but also to hinder the binding between hGH and its receptor, thereby reducing hGH signaling.
  • Such polymers may arise from proteins that do not form the appropriate tertiary structure when expressed in the target protein from the host, or may result from reduced stability of the target protein in the process.
  • such polymers are classified as abnormal peptides that are product-derived impurities, and their amount is regulated in the purity test. Accordingly, many attempts have been made to remove polymers during the production process, and size-exclusion chromatography (SEC) is generally used to separate polymers and monomers by molecular weight.
  • SEC size-exclusion chromatography
  • the target protein since the volume of the column loaded is limited (typically within 10% of the bed volume), the target protein must be concentrated at a high concentration. There is a problem.
  • the present inventors confirmed that the polymer was formed during the purification process of the sustained human growth hormone NexP-hGH, thereby separating the polymer and the monomer of the new continuous human growth hormone, which was difficult to purify by the existing method, to maintain high purity Efforts have been made to develop a method for producing monomers of the type human growth hormone NexP-hGH. As a result, anion exchange resins are used to separate the polymers and monomers of the type of sustained human growth hormone NexP-hGH from each other. It was confirmed that the monomer of hGH can be prepared and completed the present invention.
  • One object of the present invention is to provide a method for preparing monomers of NexP-hGH protein, which is a sustained human growth hormone fused with human growth hormone and alpha-1 antitrypsin variant, using anion exchange resin chromatography.
  • Another object of the present invention is to provide a monomer of NexP-hGH protein, which is a sustained human growth hormone fused to human growth hormone and alpha-1 antitrypsin variant prepared by the above method.
  • the method of the present invention has the advantage that only monomers can be obtained with a high purity of 99% or more from a mixture comprising a polymer and a monomer of the sustained human growth hormone NexP-hGH using an anion exchange resin.
  • the high purity purification method provided by the method of the present invention is applied to a production process, it is possible to purify high concentration and high purity sustained human growth hormone in large quantities, thereby increasing bioactivity and reducing antigenicity.
  • Sustained human growth hormone NexP-hGH protein can be prepared, the present invention can be usefully used for the industrial production of sustained human growth hormone.
  • FIG. 1 is a diagram showing a chromatographic separation of polymers and monomers of the sustained human growth hormone NexP-hGH using anion exchange resin chromatography.
  • FIGS. 2a and b are diagrams showing the results of measuring the purity of the sustained human growth hormone monomer by fraction in anion exchange resin chromatography by size exclusion-HPLC (SE-HPLC).
  • Figure 2a shows a high purity sustained human growth hormone monomer obtained at a salt concentration of 100mM to 250mM in anion exchange resin chromatography
  • Figure 2b is a human obtained at a salt concentration of 250mM to 350mM in anion exchange resin chromatography Purity of growth hormone monomer is shown.
  • the present invention uses anion exchange resin chromatography to prepare monomers of NexP-hGH protein, which is a sustained human growth hormone fused with human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP). Provide a method.
  • NexP-hGH protein which is a sustained human growth hormone fused with human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP).
  • the method preferably comprises the steps of: a) injecting a sample comprising a mixture of sustained human growth hormone NexP-hGH protein into a pre-equilibration anion exchange chromatography column; And b) eluting a monomer of NexP-hGH protein, which is a sustained human growth hormone, at a salt concentration gradient of 0M to 0.4M using an elution buffer having a pH of 6.0 to 9.0.
  • the sustained human growth hormone NexP-hGH protein When the sustained human growth hormone NexP-hGH protein is produced from a host cell, in addition to the monomeric sustained human growth hormone NexP-hGH protein which binds to the human growth hormone receptor and exhibits biological activity, it does not form an appropriate tertiary structure. Due to the poor stability of NexP-hGH protein or NexP-hGH protein, there is a problem in that NexP-hGH protein is produced in the form of a polymer. Therefore, when producing NexP-hGH protein from a host cell, there is a need to separate the NexP-hGH protein in high purity from the mixture containing both polymer and monomer of NexP-hGH protein, which can be used as protein medicine. It was.
  • the method of the present invention can be prepared by separating the monomer of NexP-hGH protein from the mixture of NexP-hGH protein with high purity by using anion exchange resin chromatography, the separation and purification of NexP-hGH protein in monomer form It can be useful.
  • human growth hormone is a peptide hormone, which means a hormone that can lead to human growth, cell reproduction or regeneration.
  • the human growth hormone includes any protein that can promote growth by stimulating cell differentiation of cartilage growth plates in the body.
  • the human growth hormone includes both growth hormone produced naturally and growth hormone produced using genetic engineering technology, preferably growth hormone produced using genetic engineering technology, but is not limited thereto.
  • information on the human growth hormone can be obtained from a known database such as the National Institutes of Health (NCBI) GenBank, for example, human growth hormone having an Accession Number of AAA98618, but is not limited thereto.
  • the human growth hormone can promote growth by stimulating cell differentiation of cartilage growth plates in the body, it can be usefully used as a therapeutic protein in individuals having problems in synthesis or secretion in the body.
  • alpha-1 antitrypsin variant (NexP) having an increased half-life in the body by maintaining its persistence in the body can be fused to human growth hormone, thereby being used as a form of a fusion protein (NexP-hGH) having an increased half-life.
  • NexP-hGH is a fusion protein of human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP), and may be used in combination with "persistent human growth hormone” in the present invention.
  • the NexP is a variant of alpha-1 antitrypsin (A1AT), which is a protein in the body whose half-life is increased by maintaining the persistence in the body developed by the present inventors, and is a term named by the present inventors.
  • A1AT alpha-1 antitrypsin
  • Protein sequence of alpha-1 antitrypsin, which has been deactivated by a protease inhibitor, and a manufacturing method thereof are disclosed in Korean Patent Registration No. 10-1183262 (Publication No.
  • alpha-1 antitrypsin variant that can be applied to the sustained human growth hormone NexP-hGH of the present invention is not limited to the variant disclosed in the Republic of Korea Patent Registration No. 10-1183262, the protein degradation of alpha-1 antitrypsin All mutations of certain amino acids are included for the purpose of eliminating intrinsic body activity as an enzyme inhibitor and increasing half-life.
  • the long-acting human growth hormone NexP-hGH is a protein in which the mutant of alpha-1 antitrypsin (A1AT) is fused to the N-terminus or C-terminus of human growth hormone by a gene recombinant method, compared to the first generation human growth hormone It is a substance with improved half-life in the body.
  • Most of the first generation human growth hormone drugs are manufactured in Escherichia coli, whereas the sustained human growth hormone NexP-hGH is expressed in animal cells such as CHO cells to be glycosylated. Such glycosylation has the advantage of reducing the risk of causing antigenicity in the human body by producing human growth hormone similar to the natural form.
  • the alpha-1 antitrypsin is one of proteins in mammalian blood having a molecular weight of about 50,000 Da, one of the main blood proteins having a blood concentration of about 2 mg / ml, and an alpha-1 protease inhibitor (alpha- 1 protease inhibitor).
  • Alpha-1 antitrypsin extracted from blood is marketed as emphysema under the name Prolastin under FDA approval.
  • Prolastin is commonly administered to the human body by intravenous injection at weekly intervals at a dose of 60 mg / kg and is a protein that has been demonstrated to be safe and harmful in humans.
  • the role and structure of alpha-1 antitrypsin as a protease inhibitor is well known (Elliott, P.
  • alpha-1 antitrypsin has more than 100 alleles in nature, and phenotypes are classified from A to Z according to the type of isoelectric focusing (IEF) (Stoller et al., The Lancet, 365, 2225-2236, 2005). Most of the M alleles are normal and are subdivided into various subtypes such as M1 (Val 213 ), M2, and M3 by amino acid sequence variation. Thus, alpha-1 antitrypsin used in the present invention is a specific subtype present in nature, and the same effect can be obtained with other subtypes.
  • IEF isoelectric focusing
  • alpha-1 antitrypsin protein may be obtained from a known database such as the National Institutes of Health (NCBI) GenBank, and examples thereof include an alpha-1 antitrypsin protein having an Accession Number of ABV21360 and CAJ15161, but is not limited thereto. It doesn't work.
  • NCBI National Institutes of Health
  • Such alpha-1 antitrypsin can modify one or more amino acid residues using site-directed mutagenesis to eliminate intrinsic body activity and increase half-life.
  • Variation of one or more amino acids in the alpha-1 antitrypsin variant is characterized in that the 357th amino acid Proline (P), which is the P2 position, is mutated, and more specifically, it is mutated to asparagine (N). It is done.
  • the variation of one or more amino acids in the alpha-1 antitrypsin variant includes a mutation of proline, the 357th amino acid, which is the P2 position, into an asparagine, and a variation of one or more amino acids in another position.
  • the mutation of one or more amino acids in the other position is a ninth amino acid glutamine (Glutamine, Q) to asparagine, the 232rd amino acid Cysteine (C) to Serine (Serine, S) Or the 359 th serine (Srine, S) includes all of the mutations (Threonine, T).
  • the human growth hormone NexP-hGH of the present invention which is a human growth hormone of which the half-life is increased by fusion of the alpha-1 antitrypsin variant, is obtained by introducing and expressing an expression vector containing a polynucleotide encoding the same into an animal cell. can do.
  • the obtained product containing NexP-hGH protein which is a sustained human growth hormone, may not form an appropriate tertiary structure during expression or may form a polymer in addition to the monomer due to deterioration in stability during the purification process.
  • the sustained human growth hormone in the form of a polymer may not be able to bring proper signaling in vivo or may be recognized as an antigen, and thus, it is necessary to separate monomers from the sample containing both the polymer and the monomer in high purity. In the case of using the production method of the present invention, the fraction in which the monomers are contained in high purity can be separated.
  • Step a) is a step of injecting a sample containing a mixture of the sustained human growth hormone NexP-hGH protein in a pre-equilibration anion exchange chromatography column.
  • anion exchange resin it is possible to remove other components other than the sustained human growth hormone in the sample, not only to concentrate the sustained human growth hormone monomer, but also to remove the polymer of the sustained human growth hormone. can do.
  • anion exchange chromatography refers to the separation of molecules according to their charge by binding a negatively charged (or acidic) molecule to a positively charged support.
  • the homologues of the molecules (acidic, basic and neutral) can be easily separated by this technique.
  • strong anion exchange resin and weak anion exchange resin can be used without limitation, for example, Sephadex, Sepharose, Sauce, Mono, Mini (trade name, GE healthcare), etc.
  • a resin in which the functional group of the resin is Q (Quaternary amine), DEAE (DiEthylAminoEthyl), or QAE (Quaternary Amino Ethyl) may be used.
  • the functional group of the resin may be Q or DEAE, and most preferably Q-sepharose, which is a strong anion exchange resin, may be used.
  • a BPG column filled with a Q-Sepharose resin having a functional group of Q was used (Example 3).
  • the anion exchange resin used in the anion exchange resin chromatography of the present invention may be pre-equilibration with an aqueous buffer before injecting a sample comprising a mixture of sustained human growth hormone NexP-hGH protein.
  • sample comprising a mixture of sustained human growth hormone NexP-hGH protein refers to a cell culture supernant or a cell lysate of the cells that produces the sustained human growth hormone NexP-hGH protein. cell extract), but is not limited thereto.
  • the term "partially purified” refers to a state in which a polymer or the like exists in addition to a monomer of a desired NexP-hGH protein, although at least one fractionation procedure such as chromatography is performed.
  • the partial purification process is not particularly limited in kind, and may be, for example, hydrophobic chromatography, anion exchange resin chromatography, affinity chromatography using a resin to which an antibody fragment is attached, and / or diafiltration. It is not limited.
  • the partial purification is preferably purified using at least one method selected from the group consisting of hydrophobic chromatography, anion exchange resin chromatography and affinity chromatography with a resin to which the antibody fragment is attached, more preferably a A) applying a sample comprising NexP-hGH to anion exchange resin chromatography, b) applying the eluate generated in step a) to hydrophobic resin chromatography; And c) applying the eluate generated in step b) to affinity chromatography filled with a resin to which an anti alpha-1 antitrypsin antibody fragment is attached, but is not limited thereto.
  • the solution purified by the affinity chromatography may be applied to anion exchange resin chromatography for separating the monomer of the present invention without diafiltration, but is not limited thereto, and then subjected to diafiltration and then applied to the production method of the present invention. It is possible.
  • the culture solution obtained from the transformed CHO cells is diafiltered and purified sequentially using anion exchange resin chromatography using Q-sepharose resin and hydrophobic chromatography using phenyl sepharose. Diafiltration was again performed and partially purified by an affinity chromatography process using a resin to which an anti-alpha1 antitrypsin antibody fragment was attached (Examples 1 and 2).
  • the partially purified eluate may be a sample having a final salt concentration of 0-50 mM NaCl or 0-50 mM magnesium chloride (MgCl 2 ) diluted in 0-20 mM Tris buffer at pH 6.0-9.0 without diafiltration step.
  • the present invention is not limited thereto.
  • the sample is used after dilution with NaCl of 50 mM or less or MgCl 2 or less of magnesium chloride (MgCl 2 ) without a filtration step, it has the advantage of reducing polymer loss and protein loss due to diafiltration.
  • Step b) is a step of eluting the monomer of NexP-hGH protein, a sustained human growth hormone, at a salt concentration gradient of 0M to 0.4M using an elution buffer of pH 6.0 to 9.0.
  • step b) the column into which the sample is injected is washed with a washing buffer, and eluted with a salt gradient using an elution buffer having a pH of 6.0 to 9.0.
  • the salt gradient may be a continuous salt gradient or a stepwise salt gradient, but is not limited thereto.
  • the continuous salt gradient or step salt gradient may be any one as long as the monomer of the sustained human growth hormone can be separated with high purity.
  • monomers of the sustained human growth hormone were eluted with high purity in a continuous salt gradient (Example 3).
  • a salt concentration capable of separating the monomer of the NexP-hGH protein, which is a sustained human growth hormone of the present invention may be a salt concentration of 0M to 0.4M, preferably a salt concentration of 50mM to 300mM, and more preferably 100mM. Salt concentration may be from 250 mM, but is not limited thereto.
  • the salt is preferably a sodium salt, more preferably sodium chloride, but is not limited thereto.
  • the type of buffer used in the washing and eluting step is not particularly limited, and may be, for example, sodium phosphate buffer, potassium phosphate buffer or Tris buffer, but It is not limited. However, since the polymer increases when the storage time of NexP-hGH in Tris buffer containing MgCl 2 increases, it is preferable to remove MgCl 2 in the washing and eluting steps.
  • a culture medium containing the sustained human growth hormone NexP-hGH protein was obtained from transformed CHO cells, diafiltered, and then anion exchange resin chromatography and hydrophobic chromatography were sequentially Purification was carried out. It was then diafiltered again and purified by affinity chromatography using a resin with anti-alpha1-antitrypsin antibody fragment attached.
  • An anion chromatography column (BPG column, 7 cm in diameter) using a Q-Sepharose resin was used to prepare a sample containing monomers of the sustained human growth hormone NexP-hGH protein having a purity of about 90% on SE-HPLC.
  • the 20 mM Tris buffer solution (pH 7.4) and 20 mM sodium phosphate elution buffer solution (pH 7.4) were sequentially flowed in 3 column volumes at a flow rate of 40 mL / min to remove impurities. Washed. Then, 20 mM sodium phosphate eluting solvent containing 0.4 M NaCl (pH 7.4) was used to elute the continuous human growth hormone by flowing a continuous salt concentration gradient by 10 column capacity at a flow rate of 40 ml / min.
  • the sustained human growth hormone was isolated with high purity at a NaCl concentration of 100 mM to 250 mM, while the sustained human growth hormone containing 60% or more polymers was separated at the NaCl concentration of 250 mM to 350 mM ( 1 and 2).
  • the high-purity sustained human growth hormone from which the polymer was separated in this manner did not increase the polymer by more than 1% even when concentrated at a high concentration of 12 mg / ml or more.
  • the present invention is a method capable of efficiently separating only long-acting human growth hormone NexP-hGH protein in monomer form in high purity by efficiently removing the easy-to-separate polymer form of long-acting human growth hormone NexP-hGH.
  • it can be used to separately prepare NexP-hGH with a purity of 95 to 100%.
  • the present invention provides a monomer of NexP-hGH protein, which is a sustained human growth hormone fused to human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP) prepared by the above method.
  • hGH human growth hormone
  • NexP alpha-1 antitrypsin variant
  • the human growth hormone, alpha-1 antitrypsin variant and NexP-hGH are as described above.
  • proline (P) 357 was first reacted with site directed mutagenesis.
  • An alpha-1 antitrypsin variant (A1AT (P357N)) substituted with (Asparagine, N) was prepared. Then, it was cloned with pG001-derived PCR product hGH and vector pAV1 having Xho1 and BamH1 restriction enzyme sites to prepare pT107.
  • pT107 is a P357N variant wherein hGH and A1AT are bound without a linker.
  • Clones made of the vector of ⁇ 1-1> were transduced into Chinese hamster ovary cells (CHO) and the expression of human growth hormone / alpha-1 antitrypsin mutant (107N) was confirmed.
  • Human growth hormone / alpha-1 antitrypsin fusion (107N) expressing cells were cultured in CD Opti-CHO medium at 8% CO 2 and 37 ° C. temperature, and incubated in suspension at 0.5 ⁇ 10 6 cells / ml concentration. .
  • a phenyl-sepharose loading solution was prepared by adding 4M NaCl / 20 mM sodium phosphate buffer (pH 8.0) to the Q-sepharose column eluate to give about 2.5 M NaCl / 20 mM sodium phosphate (pH 8.0). .
  • phenyl-sepharose (GE Healthcare) resin was charged to an XK-50 column (GE Healthcare), and then the column was equilibrated with sufficient flow of 20 mM sodium phosphate buffer (pH 7.5) containing 2.5 M NaCl. .
  • 20 mM sodium phosphate buffer (pH 8.0) containing 2.5 M NaCl was added at about 3 CV (column volume). Wash the column.
  • a solution containing the sustained human growth hormone NexP-hGH was recovered by flowing 20 mM sodium phosphate buffer (pH 8.0) at a flow rate of 20 ml / min at about 4 CV.
  • Resin GE Healthcare, hereinafter referred to as 'AIAT'
  • A1AT anti alpha1-antitrypsin
  • 'AIAT' XK-50 column
  • the column was equilibrated with sufficient flow of Tris buffer (pH 7.4). After about 1 L of the diafiltration solution was flowed to the prepared A1AT column at a flow rate of 20 mL / min, the column was washed again by adding Tris buffer solution (pH 7.4) containing 150 mM NaCl (pH 7.4) to about 3 CV (column volume).
  • Sustained human growth hormone NexP-hGH purified by the above method was present in more than 10% of the polymer to monomer ratio.
  • Example 3 Isolation and Purification of Sustained Human Growth Hormone NexP-hGH Polymer and Monomer by Anion Exchange Resin Chromatography
  • the purified long-acting human growth hormone NexP-hGH of Example 2 has a ratio of 10% or more of the polymer to the monomer, and it is necessary to specifically separate and purify only the monomer from the mixture in which both the monomer and the polymer are present.
  • the present inventors separated and purified only the monomer from the mixture of the polymer and monomer in the following manner.
  • NexP-hGH eluted in Example 2 was diluted 7-fold with Q column equilibration buffer, and loaded onto a BPG column (7 cm in diameter) filled with Q-Sepharose resin at a flow rate of 40 ml / min.
  • 20 mM sodium phosphate buffer (pH 7.4) was used in a three column capacity at a flow rate of 40 ml / min to wash off impurities that did not adhere to the column.
  • a sustained human growth hormone having a monomer concentration of about 98% or more is isolated at a salt concentration of about 100 mM to 250 mM, and a sustained human growth hormone comprising at least 60% of a polymer compared to the monomer at a salt concentration of 250 mM to 350 mM is isolated. It confirmed that it becomes (FIGS. 1-2).
  • high-purity sustained human growth hormone in which the polymer was separated it was confirmed that the polymer was not increased by more than 1% even when concentrated at a high concentration of 12 mg / mL or more.

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Abstract

La présente invention concerne un procédé de préparation d'un monomère de grande pureté d'une protéine NexP-hGH, qui constitue une hormone de croissance humaine à action prolongée, par chromatographie sur résine échangeuse d'anions ; et un monomère d'une protéine NexP-hGH, préparé à l'aide de ce procédé.
PCT/KR2013/010630 2012-11-21 2013-11-21 Procédé de préparation d'un nouveau monomère de l'hormone de croissance humaine à action prolongée Ceased WO2014081225A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
WO1999062936A1 (fr) * 1998-06-01 1999-12-09 Genentech, Inc. Separation des monomeres proteiques des agregats par l'utilisation de la chromatographie d'echange d'ions
US20080058507A1 (en) * 2004-02-11 2008-03-06 Hui Liu Method For The Removal Of Aggregate Proteins From Recombinant Samples Using Ion Exchange Chromatography
KR20120013359A (ko) * 2009-04-01 2012-02-14 바이오제너릭스 에이지 재조합 ｆｓｈ 정제 방법
KR101183262B1 (ko) * 2009-04-22 2012-09-14 (주)알테오젠 체내 지속성을 유지함으로 체내 반감기가 증가된 단백질 또는 펩티드 융합체, 및 이를 이용하여 체내 반감기를 증가시키는 방법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999062936A1 (fr) * 1998-06-01 1999-12-09 Genentech, Inc. Separation des monomeres proteiques des agregats par l'utilisation de la chromatographie d'echange d'ions
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