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 [IG], 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.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biophysics (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Molecular Biology (AREA)
• Biochemistry (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)
• Enzymes And Modification Thereof (AREA)

Priority Applications (7)

Applications Claiming Priority (2)

Publications (2)

Family

ID=43242573

Family Applications (1)

Country Status (12)

Cited By (3)

Families Citing this family (8)

Citations (3)

Family Cites Families (78)

• 2010
  • 2010-06-24 AU AU2010266093A patent/AU2010266093B2/en not_active Ceased
  • 2010-06-24 WO PCT/US2010/039854 patent/WO2010151688A2/en not_active Ceased
  • 2010-06-24 LT LTEP19209054.6T patent/LT3660032T/lt unknown
  • 2010-06-24 US US12/822,990 patent/US8940878B2/en active Active
  • 2010-06-24 SI SI201032164T patent/SI3660032T1/sl unknown
  • 2010-06-24 MX MX2011013417A patent/MX2011013417A/es active IP Right Grant
  • 2010-06-24 EP EP10729997.6A patent/EP2445924B2/en active Active
  • 2010-06-24 CA CA2765154A patent/CA2765154A1/en not_active Abandoned
  • 2010-06-24 FI FIEP19209054.6T patent/FI3660032T3/fi active
  • 2010-06-24 EP EP19209054.6A patent/EP3660032B1/en active Active
  • 2010-06-24 ES ES10729997T patent/ES2862580T5/es active Active
  • 2010-06-24 JP JP2012517737A patent/JP2012531428A/ja active Pending
  • 2010-06-24 DK DK19209054.6T patent/DK3660032T3/da active
  • 2010-06-24 ES ES19209054T patent/ES3056593T3/es active Active
• 2015
  • 2015-01-16 US US14/599,336 patent/US9643997B2/en active Active
• 2017
  • 2017-03-31 US US15/476,691 patent/US10577392B2/en active Active
• 2019
  • 2019-12-10 US US16/709,491 patent/US11407784B2/en active Active
• 2022
  • 2022-07-27 US US17/874,846 patent/US12312381B2/en active Active

Patent Citations (3)

Non-Patent Citations (14)

Also Published As

Similar Documents

Legal Events