WO2010151688A2 - Capture purification processes for proteins expressed in a non-mammalian system - Google Patents

Capture purification processes for proteins expressed in a non-mammalian system Download PDF

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Publication number
WO2010151688A2
WO2010151688A2 PCT/US2010/039854 US2010039854W WO2010151688A2 WO 2010151688 A2 WO2010151688 A2 WO 2010151688A2 US 2010039854 W US2010039854 W US 2010039854W WO 2010151688 A2 WO2010151688 A2 WO 2010151688A2
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WO
WIPO (PCT)
Prior art keywords
protein
separation matrix
native
solution
expressed
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/US2010/039854
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English (en)
French (fr)
Other versions
WO2010151688A3 (en
Inventor
Joseph Edward Shultz
Roger Hart
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.)
Amgen Inc
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Amgen Inc
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.)
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Publication date
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=43242573&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2010151688(A2) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to EP19209054.6A priority Critical patent/EP3660032B1/en
Priority to EP10729997.6A priority patent/EP2445924B2/en
Priority to ES10729997T priority patent/ES2862580T5/es
Priority to MX2011013417A priority patent/MX2011013417A/es
Priority to CA2765154A priority patent/CA2765154A1/en
Application filed by Amgen Inc filed Critical Amgen Inc
Priority to JP2012517737A priority patent/JP2012531428A/ja
Priority to AU2010266093A priority patent/AU2010266093B2/en
Publication of WO2010151688A2 publication Critical patent/WO2010151688A2/en
Publication of WO2010151688A3 publication Critical patent/WO2010151688A3/en
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
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • 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
    • 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
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/32—Extraction; Separation; Purification by precipitation as complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention relates generally to processes for purifying proteins expressed in non-mammalian systems in both non-native soluble and non-native insoluble forms, and more particularly to the direct capture of such proteins from a refold mixture or a cell lysate pool by a separation matrix.
  • Fc-containing proteins are typically expressed in mammalian cells, such as CHO cells.
  • mammalian cells such as CHO cells.
  • affinity chromatography to purify Fc-containing proteins is documented (see, e.g., Shukla et al, (2007) Journal of Chromatography B 848(l):28-39) and is successful, in part, due to the degree of Fc structure observed in proteins expressed in such systems.
  • Fc-containing proteins expressed in non-mammalian cells are often deposited in the expressing cells in limited solubility forms, such as inclusion bodies, that require refolding, and this has been a limiting factor in selecting non- mammalian systems for expressing Fc-containing proteins.
  • the present disclosure addresses these issues by providing simplified methods of purifying proteins comprising Fc regions that are expressed in non-mammalian expression systems in a non-native soluble form or in a non-native insoluble form.
  • This process allows the separation of proteins from cell cultures in highly concentrated pools that can be subsequently refolded at high concentrations and can be of benefit when producing large quantities of protein, particularly since the method is scalable from bench scale, which involves cultures on the order of several liters, up to production scale, which involves cultures of thousands of liters.
  • the disclosed methods can also be employed to purify proteins expressed in a non-native soluble and non-native limited solubility forms in a non-mammalian expression system that have subsequently been derivatized.
  • a protein comprising an Fc region can be associated with a small molecule, such as a toxin.
  • conjugates can be purified using the methods described herein.
  • Protein A means any protein identical or substantially similar to Staphylococcal Protein A, including commercially available and/or recombinant forms of Protein A.
  • Protein A specifically includes engineered Protein A derived media, such as Mab Select SuReTM media (GE Healthcare), in which a single subunit (e.g., the B subunit ) is replicated two or more times and joined in a contiguous sequence to form a recombinant Protein A molecule, and other non-naturally occurring Protein A molecules.
  • the disclosed method can be employed to purify a protein of interest that is expressed in a non-native soluble form in a non-mammalian cell expression system.
  • the protein of interest can be produced by living host cells that either naturally produce the protein or that have been genetically engineered to produce the protein. Methods of genetically engineering cells to produce proteins are known in the art. See, e.g. , Ausabel et al., eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods include introducing nucleic acids that encode and allow expression of the protein into living host cells.
  • a host cell will be a non-mammalian cell, such as bacterial cells, fungal cells, yeast cells, and insect cells.
  • the lysate was centrifuged in a disc-stack centrifuge to collect the protein in the solid fraction, which was expressed in a limited solubility non- native form, namely as inclusion bodies.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
PCT/US2010/039854 2009-06-25 2010-06-24 Capture purification processes for proteins expressed in a non-mammalian system Ceased WO2010151688A2 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU2010266093A AU2010266093B2 (en) 2009-06-25 2010-06-24 Capture purification processes for proteins expressed in a non-mammalian system
EP10729997.6A EP2445924B2 (en) 2009-06-25 2010-06-24 Capture purification processes for proteins expressed in a non-mammalian system
ES10729997T ES2862580T5 (es) 2009-06-25 2010-06-24 Procesos de purificación por captura para proteínas expresadas en un sistema no de mamífero
MX2011013417A MX2011013417A (es) 2009-06-25 2010-06-24 Procesos de purificacion por captura para proteinas expresadas en un sistema no mamifero.
CA2765154A CA2765154A1 (en) 2009-06-25 2010-06-24 Capture purification processes for proteins expressed in a non-mammalian system
EP19209054.6A EP3660032B1 (en) 2009-06-25 2010-06-24 Capture purification processes for proteins expressed in a non-mammalian system
JP2012517737A JP2012531428A (ja) 2009-06-25 2010-06-24 哺乳類以外の系で発現されるタンパク質の捕獲精製プロセス

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US22047709P 2009-06-25 2009-06-25
US61/220,477 2009-06-25

Publications (2)

Publication Number Publication Date
WO2010151688A2 true WO2010151688A2 (en) 2010-12-29
WO2010151688A3 WO2010151688A3 (en) 2011-05-19

Family

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Family Applications (1)

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PCT/US2010/039854 Ceased WO2010151688A2 (en) 2009-06-25 2010-06-24 Capture purification processes for proteins expressed in a non-mammalian system

Country Status (8)

Country Link
US (5) US8940878B2 (enExample)
EP (2) EP3660032B1 (enExample)
JP (1) JP2012531428A (enExample)
AU (1) AU2010266093B2 (enExample)
CA (1) CA2765154A1 (enExample)
ES (1) ES2862580T5 (enExample)
MX (1) MX2011013417A (enExample)
WO (1) WO2010151688A2 (enExample)

Cited By (3)

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WO2018018011A3 (en) * 2016-07-22 2018-03-01 Amgen Inc. Methods of purifying fc-containing proteins
US10828366B2 (en) 2015-04-22 2020-11-10 Ucb Biopharma Sprl Method of monomerisation of recombinant antibody molecules
WO2022129460A1 (en) 2020-12-18 2022-06-23 Richter Gedeon Nyrt. Methods for the purification of refolded fc-peptide fusion protein

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JP5808323B2 (ja) * 2009-06-22 2015-11-10 アムジエン・インコーポレーテツド 化学的に制御されたレドックス状態を用いたタンパク質のリフォールディング
EP3660032B1 (en) 2009-06-25 2025-10-22 Amgen Inc. Capture purification processes for proteins expressed in a non-mammalian system
PE20130557A1 (es) 2010-03-04 2013-05-19 Pfenex Inc Metodo para producir proteinas de interferon recombinantes solubles sin desnaturalizacion
US20170101435A1 (en) * 2015-10-13 2017-04-13 Therapeutic Proteins International, LLC Harvesting and perfusion apparatus
US11697670B2 (en) 2017-08-22 2023-07-11 Biogen Ma Inc. Methods for purifying antibodies having reduced high molecular weight aggregates
US11051532B2 (en) 2017-09-22 2021-07-06 Impossible Foods Inc. Methods for purifying protein
WO2020061478A2 (en) * 2018-09-21 2020-03-26 Teneobio, Inc. Methods for purifying heterodimeric, multispecific antibodies
US12011016B2 (en) 2020-09-14 2024-06-18 Impossible Foods Inc. Protein methods and compositions

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Cited By (7)

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Publication number Priority date Publication date Assignee Title
US10828366B2 (en) 2015-04-22 2020-11-10 Ucb Biopharma Sprl Method of monomerisation of recombinant antibody molecules
US11786593B2 (en) 2015-04-22 2023-10-17 UCB Biopharma SRL Method of monomerisation of recombinant antibody molecules
WO2018018011A3 (en) * 2016-07-22 2018-03-01 Amgen Inc. Methods of purifying fc-containing proteins
CN109563125A (zh) * 2016-07-22 2019-04-02 美国安进公司 含有Fc的蛋白的纯化方法
US11312745B2 (en) 2016-07-22 2022-04-26 Amgen Inc. Methods of purifying Fc-containing proteins
CN115925780A (zh) * 2016-07-22 2023-04-07 美国安进公司 含有Fc的蛋白的纯化方法
WO2022129460A1 (en) 2020-12-18 2022-06-23 Richter Gedeon Nyrt. Methods for the purification of refolded fc-peptide fusion protein

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US20200361986A1 (en) 2020-11-19
EP3660032A1 (en) 2020-06-03
CA2765154A1 (en) 2010-12-29
US11407784B2 (en) 2022-08-09
AU2010266093A1 (en) 2012-01-19
US12312381B2 (en) 2025-05-27
JP2012531428A (ja) 2012-12-10
MX2011013417A (es) 2012-03-29
AU2010266093B2 (en) 2013-06-27
EP2445924B1 (en) 2021-03-03
US10577392B2 (en) 2020-03-03
ES2862580T3 (es) 2021-10-07
US20100331526A1 (en) 2010-12-30
US20150361130A1 (en) 2015-12-17
EP2445924B2 (en) 2023-12-13
US9643997B2 (en) 2017-05-09
US8940878B2 (en) 2015-01-27
ES2862580T5 (es) 2024-06-13
EP3660032B1 (en) 2025-10-22
US20180037603A1 (en) 2018-02-08
EP2445924A2 (en) 2012-05-02
WO2010151688A3 (en) 2011-05-19
US20230016430A1 (en) 2023-01-19

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