WO2021181414A1 - Procédé de séparation d'agrégats de poids moléculaire élevé d'une protéine de fusion fc - Google Patents

Procédé de séparation d'agrégats de poids moléculaire élevé d'une protéine de fusion fc Download PDF

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Publication number
WO2021181414A1
WO2021181414A1 PCT/IN2021/050232 IN2021050232W WO2021181414A1 WO 2021181414 A1 WO2021181414 A1 WO 2021181414A1 IN 2021050232 W IN2021050232 W IN 2021050232W WO 2021181414 A1 WO2021181414 A1 WO 2021181414A1
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chromatography
composition
molecular weight
weight aggregates
fusion protein
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PCT/IN2021/050232
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English (en)
Inventor
Charles FRANKO
Shashank Mishra
Ravichandran Ramakrishnan
Gopinath GOVINDARAJAN
Krishna Prasad Chellapilla
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Dr. Reddy’S Laboratories Limited
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Publication of WO2021181414A1 publication Critical patent/WO2021181414A1/fr

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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • 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
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to protein purification methods.
  • disclosed is a method for purifying fusion proteins using chromatography.
  • Fc-fusion proteins are bioengineered polypeptides that join the crystallizable fragment (Fc) domain of an antibody with another biologically active protein domain to generate a molecule with unique structure-function properties and significant therapeutic potential.
  • the gamma immunoglobulin (IgG) isotype is often used as the basis for generating Fc-fusion proteins because of favorable characteristics such as recruitment of effector function and increased plasma half-life.
  • Fc-fusion proteins Given the range of proteins that can be used as fusion partners, Fc-fusion proteins have numerous biological and pharmaceutical applications, which has launched Fc- fusion proteins into the forefront of drug development.
  • Fc-fusion proteins can be commercially manufactured using platform upstream and downstream methods based for (monoclonal antibodies) mAbs.
  • Fc-fusion proteins receptor domains generally contain one or more glycosylation sites (both N- and O-linked) in contrast to single glycosylation site for mAbs.
  • the oligosaccharide structures are more varied and complex (complex and high mannose; bi-, tri- and tetra-antennary) in their receptor domains than IgG Fc (complex, bi-antennary) and can contain more sialic acid residues. The latter can shift the pi of Fc-fusion proteins into an acidic pH range and impart significantly more charge heterogeneity on them than that of the conventional mAbs.
  • there are unique attributes of Fc-Fusion proteins that would require optimization or redesigning of the commercial process used for the manufacture of mAbs.
  • Chromatography is widely used and has become indispensable in purification of any protein, including Fc-fusion proteins.
  • Combinations of different chromatographic techniques such as affinity chromatography, ion-exchange chromatography, size-exclusion chromatography, hydrophobic interaction chromatography are generally employed for achieving high level of purity and removal of contaminants.
  • several physicochemical conditions such as pH, temperature, buffer components and concentration, salt, chromatographic support, binding ligand, properties of the protein, such as hydrophobicity, need to the considered so as to have the protein in pure yet stable and functional form.
  • an Fc-fusion protein is abatacept, which exists as a covalent homodimer linked through an inter-chain disulfide bond.
  • the linkage between the two monomers can, however, also be of non-covalent nature. This non-covalent interaction between two monomers can result in the formation of dimers which are unstable and hence should be removed from the purified composition of abatacept.
  • the objective of the present invention is to purify Fc-fusion protein from contaminants, such as high molecular weight (HMW) aggregates and non-covalent dimers arising from the cell culture process, using chromatography.
  • contaminants such as high molecular weight (HMW) aggregates and non-covalent dimers arising from the cell culture process, using chromatography.
  • the present invention discloses a method for purifying an Fc-Fusion protein from the contaminants, preferably high-molecular weight aggregates and non-covalently linked dimers, the method comprises use of hydrophobic interaction chromatography (HIC).
  • HIC hydrophobic interaction chromatography
  • the method discloses the use of hydrophobic interaction chromatography in flow through mode using a linear butyl ligand for purifying an Fc-fusion protein composition from contaminants, preferably high-molecular weight aggregates and non-covalent dimers, thus obtaining a purified composition.
  • the inventive method discloses a HIC support / stationary phase with a linear butyl ligand for the purification of CTLA4-Ig fusion protein from its HMW aggregates and non-covalent dimers.
  • the method as disclosed herein is successful in a significant reduction of HMW aggregates, i.e., greater than 90% reduction in aggregates and 100% removal of non-covalent dimers are achieved by the method.
  • the method disclosed in the invention is capable of being used at a commercial scale for controlling the level of HMW aggregates and non-covalent dimers and thus, in the manufacture of a purified composition of the said CTLA4-Ig fusion protein.
  • Figure 1 shows the chromatography process flow chart for the evaluation of hexyl, octyl, phenyl and butyl ligands in flow-through mode for the development of the HIC step.
  • Figure 2 shows the chromatography process flow chart for the evaluation of phenyl ligand in bind and elute mode for the development of the HIC step.
  • Figure 3 shows the chromatography process flow chart for optimization of the HIC step using butyl ligand in flow-through mode.
  • Figure 4 shows the chromatography process flow chart for the optimized HIC step using butyl ligand.
  • Fc fusion protein is a protein that contains an Fc region of an immunoglobulin is fused or linked to a heterologous polypeptide.
  • the heterologous polypeptide fused to the Fc region may be a polypeptide from a protein other than an immunoglobulin protein.
  • the heterologous polypeptide may be a ligand polypeptide, a receptor polypeptide, a hormone, cytokine, growth factor, an enzyme, or other polypeptide that is not a component of an immunoglobulin.
  • Such Fc fusion proteins may comprise an Fc region fused to a receptor or fragment thereof or a ligand from a receptor including, but not limited to, any one of the following receptors: both forms of TNFR (referred to as p55 and p75), Interleukin-1 receptors types I and II (as described in EP Patent No. 0460846, US Patent No. 4,968,607, and US Patent No. 5,767,064, which are incorporated by reference herein in their entirety), Interleukin-2 receptor, Interleukin-4 receptor (as described in EP Patent No. 0 367 566 and US Patent No.
  • Interleukin- 15 receptor Interleukin- 17 receptor
  • Interleukin- 18 receptor granulocyte- macrophage colony stimulating factor receptor
  • granulocyte colony stimulating factor receptor receptors for oncostatin-M and leukemia inhibitory factor
  • receptor activator of NF-kappa B RNK, as described in US Patent No. 6,271,349, which is incorporated by reference herein in its entirety
  • VEGF receptors EGF receptor
  • FGF receptors receptors for TRAIL (including TRAIL receptors 1,2,3, and 4), and receptors that comprise death domains, such as Fas or Apoptosis- Inducing Receptor (AIR).
  • Fc fusion proteins also include peptibodies, such as those described in WO 2000/24782, which is hereby incorporated by reference in its entirety.
  • CTLA4-Ig Fc-fusion refers to a protein that links the extracellular domain of human cytotoxic T-lymphocyte associated antigen 4 (CTLA4) to the modified Fc (hinge, CH2 and CH3 domains) region of human immunoglobulin G1. It is a homodimer of two polypeptide chains connected together through one disulfide bond in the CTLA4 domain.
  • “High molecular weight aggregates” as referred herein encompasses association of at least two molecules of a product of interest, e.g., Fc-Fusion protein.
  • the association of at least two molecules of a product of interest may arise by any means including, but not limited to, non- covalent interactions such as, e.g., charge-charge, hydrophobic and van der Waals interactions; and covalent interactions such as, e.g., disulfide interaction or nonreducible crosslinking.
  • An aggregate can be a trimer, tetramer, or a multimer greater than a tetramer, etc.
  • protein aggregates includes any higher order species of the Fc-containing protein.
  • Non-covalent dimer refers to the two monomeric polypeptides chains of abatacept connected together by non-covalent interactions.
  • the composition may be "partially purified” (i.e., having been subjected to one or more purification steps) or may be obtained directly from a host cell or organism producing the antibody (e.g., the composition may comprise harvested cell culture fluid).
  • load herein refers to the composition loaded onto the chromatography material, i.e., HIC support or mixed mode chromatography (MMC) support.
  • the chromatography material is equilibrated with an equilibration buffer prior to loading the composition which is to be purified.
  • Mated Mode Chromatography refers to a form of chromatography that uses a chromatographic support with at least two unique types of functional groups, each interacting with the molecule or protein of interest.
  • Mixed mode chromatography generally uses ligands that have more than one type of interaction with target proteins and/or impurities. For example, a charge-charge type of interaction and/or a hydrophobic or hydrophilic type of interaction, or an electroreceptor-donor type interaction. In general, based on the difference in the total interaction, the target protein and one or more impurities can be separated under various conditions.
  • flow-through mode refers to that process wherein the target protein is not bound to the chromatographic support but instead obtained in the unbound or “flow- through” fraction during loading or post load wash of the chromatography support.
  • Aggregate concentration can be measured in a protein sample using Size Exclusion Chromatography (SEC), a well-known and widely accepted method in the art.
  • Size exclusion chromatography uses a molecular sieving retention mechanism, based on differences in the hydrodynamic radii or differences in size of proteins. Large molecular weight aggregates cannot penetrate or only partially penetrate the pores of the stationary phase. Hence, the larger aggregates elute first and smaller molecules elute later, the order of elution being a function of the size.
  • the present invention discloses a method for purifying the Fc-Fusion proteins from the contaminants, the method comprises use of hydrophobic interaction chromatography.
  • the method is used to obtain a purified composition of a CTLA4-Ig fusion protein using hydrophobic interaction chromatography.
  • the method is used to obtain a purified composition of a CTLA4-Ig fusion protein by hydrophobic interaction chromatography, wherein the chromatographic medium has a straight butyl chain ligand attached to a hydrophilic base matrix.
  • the method is used to obtain a purified composition of a CTLA4- Ig fusion protein by hydrophobic interaction chromatography, wherein the chromatographic support has a straight butyl chain ligand attached to a hydrophilic base matrix and the chromatography is operated in flow-through mode.
  • the method is used to obtain a purified composition of a CTLA4- Ig fusion protein, the method comprising steps of hydrophobic interaction chromatography and mixed-mode chromatography, wherein the mixed mode chromatography follows the hydrophobic interaction chromatography.
  • the method is used to obtain a purified composition of a CTLA4- Ig fusion protein, the method comprising steps of hydrophobic interaction chromatography and mixed-mode chromatography, wherein the mixed mode chromatography follows the hydrophobic interaction chromatography and wherein both the chromatographic steps are operated in flow-through mode.
  • the method is used to obtain a purified composition of a CTLA4-Ig fusion protein, the method comprising steps of hydrophobic interaction chromatography and mixed-mode chromatography, wherein the mixed mode chromatography follows the hydrophobic interaction chromatography, and wherein both the chromatographic steps are operated in flow-through mode, and wherein the HIC chromatographic support has a straight butyl chain ligand attached to a hydrophilic base matrix.
  • the method is capable of significant removal of HMW aggregates and non-covalent dimers.
  • greater than 90% reduction in the HMW aggregates level and 100% removal of non-covalent dimers are achieved by the method.
  • the method is used to obtain a purified composition of a CTLA-Ig fusion protein, the method comprising hydrophobic interaction chromatography, wherein the HMW aggregate level is ⁇ 1.5% and non-covalent dimers are below detectable limit (i.e., complete removal) in fusion protein composition obtained as the flow-through of the hydrophobic interaction chromatography.
  • the method is used to obtain a purified composition of a CTLA-Ig fusion protein, the method comprising hydrophobic interaction chromatography and mixed mode chromatography, wherein the HMW aggregate level is ⁇ 1.0% and non-covalent dimers are below detectable limit (i.e., complete removal) in fusion protein composition obtained as the flow-through of the mixed-mode chromatography.
  • the chromatographic medium used in hydrophobic interaction chromatography is preferably CaptoTM Butyl.
  • the buffer used for hydrophobic interaction chromatography is Tris buffer and the salt used is ammonium sulphate.
  • the buffer solution used for mixed-mode chromatography is phosphate buffer.
  • the method comprises use of one or more chromatographic steps before HIC chromatography, wherein the preceding chromatography does not comprise an ion exchange chromatography step.
  • the polishing steps may be selected from ion exchange chromatography, hydrophobic interaction chromatography, hydrophobic charge induction chromatography, mixed-mode chromatography.
  • the method comprises use of one or more polishing chromatography steps after HIC chromatography.
  • the polishing steps may be selected from ion exchange chromatography, hydrophobic ion exchange chromatography, hydrophobic charge induction chromatography, mixed-mode chromatography.
  • the method employs use of one or more steps such as viral inactivation, filtration and diafiltration. These steps may be interspersed between the chromatographic steps or the after all the chromatographic steps.
  • the purity of the fusion protein composition is more than 95%.
  • the fusion protein is CTLA4-Ig fusion protein.
  • the fusion protein is abatacept.
  • CTLA4-IgG fusion protein was cloned and expressed in a Chinese Hamster Ovary cell line and the cell culture broth containing the expressed fusion protein was harvested, clarified and subjected to protein- A affinity chromatography.
  • the eluate from protein- A affinity chromatography was subjected to low-pH incubation and depth filtration, and the eluate was loaded onto an Octyl ligand. Details of the salts used for evaluation are listed in Table 1. Column chromatography conditions are listed in Table 2 and buffer details are captured in Table 3.
  • the loading and post-load washing steps were fractionated 1 column volume (CV) each, and representative pools were analysed by SEC-UPLC for non-covalent dimer content and SEC-HPLC for HMW aggregate content.
  • able 1 Salts used for evaluation of octyl ligand
  • Table 4 shows the %HMW and %non-covalent dimer data for HIC load and HIC flow through evaluation
  • CTLA4-IgG fusion protein was cloned and expressed in a Chinese Hamster Ovary cell line and the cell culture broth containing the expressed fusion protein was harvested, clarified and subjected to protein- A affinity chromatography.
  • the eluate from protein-A affinity chromatography was subjected to low-pH incubation and depth filtration, and the eluate was loaded onto a Hexyl ligand. Details of the salts used for evaluation are listed in Table 5.
  • Column chromatography conditions are listed in Table 6 and buffer details are captured in Table 7.
  • the loading and post-load washing steps were fractionated 1 CV each, and representative pools were analysed by SEC-UPLC for non-covalent dimer content and SEC-HPLC for HMW aggregate content.
  • CTLA4-IgG fusion protein was cloned and expressed in a Chinese Hamster Ovary cell line and the cell culture broth containing the expressed fusion protein was harvested, clarified and subjected to protein- A affinity chromatography.
  • the eluate from protein- A affinity chromatography was subjected to low-pH incubation and depth filtration, and the eluate was loaded onto a Phenyl ligand. Details of the salts used for evaluation are listed in Table 9.
  • Column chromatography conditions are listed in Table 10 and buffer details are captured in Table 11.
  • the loading and post-load washing steps were fractionated 1 CV each, and representative pools were analysed by SEC-UPLC for non-covalent dimer content and SEC- HPLC for HMW aggregate content.
  • Table 10 Chromatography conditions used for the evaluation of phenyl ligand in bind and elute mode
  • Table 11 Buffer details for phenyl ligand evaluation in bind and elute mode ligand evaluation in bind and elute mode
  • CTLA4-IgG fusion protein was cloned and expressed in a Chinese Hamster Ovary cell line and the cell culture broth containing the expressed fusion protein was harvested, clarified and subjected to protein- A affinity chromatography.
  • the eluate from protein-A affinity chromatography was subjected to low-pH incubation and depth filtration, and the eluate was loaded onto a Phenyl ligand. Details of the salts used for evaluation are listed in Table 13. Column chromatography conditions are listed in Table 14 and buffer details are captured in Table 15.
  • the loading and post-load washing steps were fractionated 1 CV each, and representative pools were analysed by SEC-UPLC for non-covalent dimer content and SEC- HPLC for HMW aggregate content.
  • Table 13 Salt used for evaluation of phenyl ligand in flow-through mode
  • Table 14 Chromatography conditions used for the evaluation of phenyl ligand in flow-through mode
  • Example 5 Evaluation of Butyl ligand in Flow-through mode CTLA4-IgG fusion protein was cloned and expressed in a Chinese Hamster Ovary cell line and the cell culture broth containing the expressed fusion protein was harvested, clarified and subjected to protein- A affinity chromatography. The eluate from protein-A affinity chromatography was subjected to low-pH incubation and depth filtration, and the eluate was loaded onto a Butyl ligand. Details of the salts used for evaluation are listed in Table 17. Column chromatography conditions are listed in Table 18 and buffer details are captured in Table 19. The loading and post-load washing steps were fractionated 1 CV each, and representative pools were analysed by SEC-UPLC for non-covalent dimer content and SEC- HPLC for HMW aggregate content.
  • CTLA4-IgG fusion protein was cloned and expressed in a Chinese Hamster Ovary cell line and the cell culture broth containing the expressed fusion protein was harvested, clarified and subjected to protein- A affinity chromatography.
  • the eluate from protein-A affinity chromatography was subjected to low-pH incubation and depth filtration, and the eluate was loaded onto a Butyl ligand. Details of the salts used for evaluation are listed in Table 21.
  • Column chromatography conditions are listed in Table 22 and buffer details are captured in Table 23.
  • the loading and post-load washing steps were fractionated 1 CV each, and representative pools were analysed by SEC-UPLC for non-covalent dimer content and SEC- HPLC for HMW aggregate content.
  • Table 22 Chromatography conditions used for the optimization of HIC step using Butyl ligand in flow-through mode
  • Table 23 Buffer details for optimization of HIC step using butyl ligand in flow-through mode optimization of HIC step using Butyl ligand in flow-through mode
  • Example 7 Scale-up of HIC step using Butyl ligand
  • CTLA4-IgG fusion protein was cloned and expressed in a Chinese Hamster Ovary cell line and the cell culture broth containing the expressed fusion protein was harvested, clarified and subjected to protein- A affinity chromatography.
  • the eluate from protein-A affinity chromatography was subjected to low-pH incubation and depth filtration, and the eluate was loaded onto a Butyl ligand. Column chromatography conditions are listed in Table 25 and buffer details are captured in Table 26.
  • Table 25 Chromatography conditions used for the scale-up of HIC step using Butyl ligand in flow through mode
  • Table 27 non-covalent dimer data for HIC load and HIC flow-through for scale-up of HIC step using butyl ligand in flow-through mode
  • the HIC flow-through fraction comprising the protein of interest was loaded onto the CHT support after diafiltration in 10 mM phosphate buffer comprising 15 mM NaCl, pH 7.5.
  • Mixed mode chromatography was operated in flow-through mode. The chromatographic details are listed in Table 28 and the %HMW and %non-covalent dimer data are shown in Table 29.

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Abstract

La présente invention concerne un procédé de purification de protéine de fusion par rapport à des contaminants. Le procédé décrit concerne un procédé de réduction d'agrégats de poids moléculaire élevé et de dimères non covalents d'une protéine de fusion Fc par chromatographie d'interaction hydrophobe sans étape de chromatographie d'échange d'anions préalable. Le procédé décrit également l'utilisation de la chromatographie en mode mixte pour la réduction d'agrégats de poids moléculaire élevé de la protéine de fusion Fc.
PCT/IN2021/050232 2020-03-11 2021-03-10 Procédé de séparation d'agrégats de poids moléculaire élevé d'une protéine de fusion fc WO2021181414A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008087184A2 (fr) * 2007-01-17 2008-07-24 Merck Serono S.A. Procédé pour la purification de protéines contenant fc
WO2009053358A1 (fr) * 2007-10-22 2009-04-30 Merck Serono S.A. Procédé de purification de protéines de fusion avec fc
WO2009111347A1 (fr) * 2008-02-29 2009-09-11 Biogen Idec Ma Inc. Protéines hybrides purifiées d’immunoglobuline et leurs procédés de purification

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008087184A2 (fr) * 2007-01-17 2008-07-24 Merck Serono S.A. Procédé pour la purification de protéines contenant fc
WO2009053358A1 (fr) * 2007-10-22 2009-04-30 Merck Serono S.A. Procédé de purification de protéines de fusion avec fc
WO2009111347A1 (fr) * 2008-02-29 2009-09-11 Biogen Idec Ma Inc. Protéines hybrides purifiées d’immunoglobuline et leurs procédés de purification

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