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WO2021176311A1 - Procédé de purification amélioré de ranibizumab - Google Patents

Procédé de purification amélioré de ranibizumab Download PDF

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Publication number
WO2021176311A1
WO2021176311A1 PCT/IB2021/051601 IB2021051601W WO2021176311A1 WO 2021176311 A1 WO2021176311 A1 WO 2021176311A1 IB 2021051601 W IB2021051601 W IB 2021051601W WO 2021176311 A1 WO2021176311 A1 WO 2021176311A1
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WO
WIPO (PCT)
Prior art keywords
ranibizumab
buffer
cysteinylated
antibody
conductivity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2021/051601
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English (en)
Inventor
Sandeep Suresh Somani
Ashok Kumar Mishra
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lupin Ltd
Original Assignee
Lupin Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lupin Ltd filed Critical Lupin Ltd
Publication of WO2021176311A1 publication Critical patent/WO2021176311A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'

Definitions

  • the present invention relates to protein purification.
  • the invention is related to the separation of ranibizumab from cysteinylated ranibizumab. Background of the invention
  • Recombinant DNA technology methods for production of therapeutic proteins, generally adopt mammalian or bacterial based expression systems.
  • mammalian based expression systems have experienced a major boost with the advent of antibody-based therapeutics, however bacteria based expression systems continue to be widely used for production of non-glycosylated peptides and proteins.
  • the bacterial expression systems are easy to manipulate, less time consuming and higher in yield.
  • recombinant proteins are mostly expressed as inclusion bodies. Recovery of the therapeutically active proteins from the inclusion bodies involves multiple steps such as unfolding and refolding of the protein using harsh conditions in presence of chaotropic agents and reducing thiols.
  • cysteine residues in protein for example antibody, IgG, Fab fragments
  • cysteine residues in protein which do not have any role in disulfide bond formation
  • cysteine residues which do not have any role in disulfide bond formation can, form a disulfide bond with free cysteine or glutathione present in refolding buffer or solution. This process is known as cysteinylation (cysteine) or glutathionylation (glutathione). Cysteinylated/glutathionylated proteins are the part of product related impurities.
  • Ranibizumab is a monoclonal antibody fragment (Fab) which lacks an Fc region.
  • the molecular weight of ranibizumab is approximately 48 kDa (23 kDa and 25 kDa for the light and heavy chain, respectively).
  • Ranibizumab contains 10 cysteine residues forming 4 intra-chain and 1 inter-chain disulfide bonds.
  • Ranibizumab is commercially available under the brand name Lucentis®. Lucentis® is used to treat the "wet form" of age-related macular degeneration and swelling in the retina caused by diabetes or by a blockage in the blood vessels.
  • Ranibizumab binds to human vascular endothelial growth factor A (VEGF-A) and inhibits the biologic activity of VEGF-A.
  • VEGF-A human vascular endothelial growth factor A
  • Ranibizumab is produced by an E. coli expression system.
  • WO2012/013682 discloses a method for purification of Fab fragment using cation exchange chromatography followed by anion exchange chromatography.
  • US8044017 and US8710196 describe the importance of changing conductivities for separation of polypeptides.
  • W02013/067301 discloses an overloading method for protein purification in which the protein load amount is more than the dynamic binding capacity of the column.
  • WO1999/062936 discloses purification of monomeric protein up to 99.5% purity using ion exchange chromatography using salt gradient.
  • Protein purification methods are mostly governed by the type of protein, method for production, quantity and quality of impurities. Sometimes, even after two or more chromatographic purification steps some impurities are still present in the protein. Purification of any specific protein requires a novel solutions or approaches and these tasks become more challenging and unpredictable, when the protein of interest and the impurities demonstrate similar or proximal physiochemical properties, for example when both, the impurity and the protein of interest, are acidic or are basic or share similar hydrophobicity etc.
  • the main aim of the instant invention is to develop a cost effective and efficient process for removal of product related impurities such as cysteinylated or glutathionylated from a protein mixture during production of therapeutic protein.
  • the instant invention provides a separation method of ranibizumab from cysteinylated ranibizumab.
  • the invention provides an efficient and economic process for the purification of antibody or antibody’s fragment with free or unpaired cysteine residues from product related impurities.
  • the instant invention provides a method for purifying antibody or antibody’s fragment from a composition comprising the antibody or antibody’s fragment and contaminants wherein the contaminants are product related impurities.
  • the product related impurities are cysteinylated or glutathionylated antibody or fragment thereof.
  • the invention describes production of heavy chain and light chain of antibody or its fragment thereof in bacteria in the form of inclusion bodies, solubilizing the heavy chain and light chain, refolding the heavy chain and light chain under suitable conditions to obtain refolded antibody or its fragment thereof, wherein solubilization or refolding buffer comprises cysteine or glutathione and purifying the refolded protein using ion exchange chromatography to remove cysteinylated antibody or fragment thereof.
  • the ion exchange chromatography is anion exchange chromatography.
  • the invention provides a large-scale separation method of antibody or fragment thereof from antibody or fragment thereof, the method comprises;
  • wash buffer is on second pH and second conductivity
  • the antibody or its fragment thereof expressed in bacteria is Fab fragment.
  • the Fab fragment is ranibizumab.
  • the invention provides a separation method of ranibizumab from cysteinylated ranibizumab, the method comprises, loading the composition comprising ranibizumab and cysteinylated ranibizumab onto anion exchange chromatography material, and eluting the ranibizumab, wherein the eluted ranibizumab has reduced amount of cysteinylated ranibizumab.
  • the invention provides a large-scale separation method of ranibizumab from cysteinylated ranibizumab, the method comprises; (a) loading the composition comprising ranibizumab and cysteinylated ranibizumab onto anion exchange resin using load buffer wherein the load buffer is at first pH and first conductivity, (b) washing the anion exchange resin with wash buffer wherein the wash buffer is on second pH and second conductivity,
  • the first pH is greater than the third pH and first conductivity is lower than the third conductivity.
  • first pH and first conductivity are essentially the same as second pH and second conductivity.
  • the pH gradient comprises a decrease in pH from about pH 9 to about pH 8.
  • the pH gradient is generated using one or more buffer solutions.
  • one or more buffers are Tris-Cl, phosphate, or CAPS.
  • the loading buffer, wash buffer and elution buffer concentration are in the range of about 10mm to about lOOmM.
  • the loading buffer, wash buffer and elution buffer pH are in the range of about 8.0 to about 10.
  • the loading buffer, wash buffer and elution buffer conductivities are in the range of about O.lmS/cm to about 5 mS/cm.
  • the invention provides separation method of ranibizumab from cysteinylated ranibizumab the method comprises: (a) loading the composition comprising ranibizumab and cysteinylated ranibizumab onto anion exchange resin using loading buffer comprising, pH about 9 to about 10, and conductivity about 0.5 to about 5 mS/cm,
  • the protein load on anion exchnage chromatography is about 1 mg/ml of resin to about 10 mg/ml of resin.
  • separation of ranibizumab from cysteinylated ranibizumab using anion exchange chromatography step may be applied after removal of other process and product related impurities such as host cell impurities or aggregates.
  • the eluted ranibizumab may be further purified using one or more chromatographic step.
  • Figure 1 Illustrates the chromatogram of Anion exchange chromatography carried out in linear gradient mode.
  • FIG. 2 Reversed-phase HPLC chromatograms of ranibizumab after anion exchange chromatography: Cysteinylated (Fab-Cys fragments) and non- cysteinylated (Fab) are separated.
  • FIG. 3 Reversed-phase HPLC chromatograms of ranibizumab after anion exchange chromatography: AEX eluate devoid of Cysteinylated Fab/Fab fragments.
  • the instant invention relates to purification of ranibizumab in linear gradient mode for separation of cysteinylated ranibizumab from target ranibizumab protein.
  • the anion exchange chromatography is carried out in bind-elute mode wherein the ranibizumab and cysteinylated ranibizumab binds with ion exchange chromatographic column and linear pH gradient is applied to elute ranibizumab before to cysteinylated ranibizumab
  • purifying or “purified” or “separating,” or “isolating,” or purification” or “separation” as used interchangeably herein, refer to increasing the degree of purity of a polypeptide or protein of interest or a target protein from a protein mixture comprising the polypeptide and one or more impurities or contaminants including at least one of the product related impurities.
  • antibody or fragment thereof is used in the broadest sense and covers monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies, and single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab')2, and Fv).
  • buffer refers to a solution used for solubilization of inclusion bodies.
  • refolding buffef or “refolding solution” refers to a solution used for recovering a desire refolded protein confirmation from solubilized inclusion bodies.
  • binding-elute mode refers to a mode of purification by chromatography, wherein the Protein-of-interest when loaded on the column is bound to the chromatographic resin and is subsequently eluted with an elution buffer.
  • the term “about”, as used herein, is intended to refer to ranges of approximately 10- 20% greater than or less than the referenced value. In certain circumstances, one of skill in the art will recognize that, due to the nature of the referenced value, the term “about” can mean more or less than a 10-20% deviation from that value.
  • biologically active refers to a molecule with a biologically active conformation and perform desire biological activities.
  • Anion exchange resin or “Anion exchange chromatography” mentioned in the embodiments refers to a solid chromatographic support, which has a positively charged ligand such as a quaternary amino group attached thereto, capable of ionic interaction with a negatively charged protein or a functional group under suitable conditions.
  • the anion exchange resin can be any weak or strong anion exchange resin or a membrane, which could function as a weak or a strong anion exchanger.
  • anion exchange resins include without any limitation DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX Sepharose Fast Flow, Q Sepharose high Performance, QAE SEPHADEX and FAST Q SEPHAROSE from GE Healthcare, Macro-Prep DEAE and Macro-Prep High Q from Biorad, Q-Ceramic Hyper D, DEAE-Ceramic Hyper D, from Pall Corporation.
  • gradient elution refer generally to conditions in which pH and/or conductivity is either increased or decreased using at least two buffers wherein the buffers are different in terms of pH or conductivity or both.
  • linear gradient is used here to refer to conditions in which pH and/or conductivity is either increased or decreased gradually using at least two buffers wherein the buffers are different in terms of pH or conductivity or both.
  • a molarity of a load buffer is essentially the same as wash buffer means the difference between molarity is not significant. Most preferably the essentially same is used when both molarity are same.
  • impurity refers to any proteinaceous or non-proteinaceous molecular entity distinct from the than the protein of interest.
  • the impurity includes, without limitation: host cell DNA, fragment, aggregate, another polypeptide, endotoxin, bacterial cell culture media component etc.
  • the protein of interest may be described as the fraction of protein, substantially free of impurities, as described herein, and can be obtained as part of the flow-through from the anion exchange chromatography
  • cystylation or “glutathionylation” refers to undesirable posttranslational modification in pharmaceutical proteins, which may lead to a conformational isoform with undesirable properties, such as low binding, low biological activity and low stability.
  • cysteine of “protein” is involved in free disulfide bond formation
  • cystylation or “glutathionylation” involve the formation of di-sulfide bond with free cysteine of glutatheon respectively.
  • cystysteinylated or “glutohionylated” refers to protein with free disulfide bond formation.
  • the protein is ranibizumab.
  • cysteinylated ranibizumab refers to ranibizumab wherein either heavy or light chain or both chains of ranibizumab are cysteinylated.
  • W02017/029620 Solubilization and refolding of ranibizumab has already been disclosed in W02017/029620.
  • W02017/029620 describe a method for production of active ranibizumab, which is incorporated herein by reference.
  • IBs Inclusion bodies
  • refolding buffer comprised of 0.6 M Arginine, 5% sorbitol, 2-10 mM of cystine and 50 mM Tris at pH 10 and temperature of 5 -10 °C within 1 h at constant flow rate. Refolding was carried out for further 14-24 hrs. The refolded ranibizumab was passed through ultrafiltration step to remove refolding buffer component and diafiltration to change the buffer for anion exchange chromatography.
  • the anion exchange chromatography was carried out using Fractogel® (Millipore) TMAE resin.
  • TMAE resin beads having positive charge will bind the ranibizumab target protein which is having net negative charge at operating pH of chromatography. This step is mainly used for removal of product related impurities.
  • Anion exchange chromatography was carried out as follows.
  • the pH of antibody composition comprising ranibizumab and cysteinylated ranibizumab was adjusted to 9.10 ⁇ 0.15 and conductivity was adjusted to ⁇ 1.0 mS/cm.
  • the column was equilibrated with 3-5 CVs of equilibration buffer (50 mM Tris, pH 9.1, conductivity 0.6 mS/cm).
  • the antibody composition comprising ranibizumab and cysteinylated ranibizumab was loaded on anion exchange resin.
  • the antibody load was about 7mg/ml of resin.
  • elution buffer 50 mM Tris, pH- 8.0, conductivity 2.4 mS/cm in linear gradient mode of 0-100% (i.e. equilibration buffer and elution buffer) in 20 CV and elution peak was collected.
  • Figure 1 The details of the chromatogram of anion exchange chromatography is provide in Figure 1.
  • Figure 1 showed the presence of protein of interest and post elutes peaks. Both peaks were collected and characterized using reverse phase HPLC.
  • the post eluted peak obtained in anion exchange chromatography were characterized using reverse phase HPLC ( Agilent 1260 HPLC with MWD and FLD) and column Agilent Zorbax 300 SB - C8, 150 X 4.6 mm, 5m, Catalogue No.:883995-906 ) using the protocol as suggested by the instrument provider.
  • the results of RP-HPLC are furnished in Figure 2 & Figure 3.
  • the post-eluted peak were identified as cysteinylated Heavy chain and light chain of ranibizumab by LC- MS.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un procédé rentable et efficace pour la séparation d'impuretés cystéinylées ou glutathionylées d'un mélange protéique thérapeutique pendant la production d'une protéine thérapeutique. En particulier, la présente invention concerne un procédé de séparation de ranibizumab à partir de ranibizumab cystéinylé à l'aide d'une chromatographie par échange d'anions.
PCT/IB2021/051601 2020-03-05 2021-02-26 Procédé de purification amélioré de ranibizumab Ceased WO2021176311A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN202021009533 2020-03-05
IN202021009533 2020-03-05

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WO2021176311A1 true WO2021176311A1 (fr) 2021-09-10

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109369806A (zh) * 2019-01-14 2019-02-22 迈威(上海)生物科技有限公司 苏金单抗制品中半胱氨酸化变异体的去除方法
US20190135909A1 (en) * 2010-07-27 2019-05-09 Ucb Pharma, S.A. Protein purification

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190135909A1 (en) * 2010-07-27 2019-05-09 Ucb Pharma, S.A. Protein purification
CN109369806A (zh) * 2019-01-14 2019-02-22 迈威(上海)生物科技有限公司 苏金单抗制品中半胱氨酸化变异体的去除方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DOUGLAS BANKS ET AL.: "Removal of Cysteinylation from an Unpaired Sulfhydryl in the Variable Region of a Recombinant Monoclonal IgG1 Antibody Improves Homogeneity, Stability, and Biological Activity", JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 97, no. 2, February 2008 (2008-02-01), pages 764 - 779, XP002545264 *

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