US008940878B2
`
`(12) United States Patent
`Shultz et a1.
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 8,940,878 B2
`*Jan. 27, 2015
`
`(54) CAPTURE PURIFICATION PROCESSES FOR
`PROTEINS EXPRESSED IN A
`NON-MAMMALIAN SYSTEM
`
`(75) Inventors: Joseph Edward Shultz, Santa Rosa
`Valley, CA (US); Roger Hart, Loveland,
`CO (U S)
`
`(73) Assignee: Amgen Inc., Thousand Oaks, CA (US)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 471 days.
`This patent is subject to a terminal dis
`claimer.
`
`(21) Appl.No.: 12/s22,990
`
`(22) Filed:
`
`Jun. 24, 2010
`
`(65)
`
`Prior Publication Data
`
`US 2010/0331526 A1
`
`Dec. 30, 2010
`
`Related US. Application Data
`
`(60) Provisional application No. 61/220,477, ?led on Jun.
`25, 2009.
`
`(51) Int. Cl.
`co 7K 1/22
`co 7K 1/14
`co 7K 1/18
`co 7K 1/32
`(52) vs. C].
`CPC . C07K1/22 (2013.01); C07K1/145 (2013.01);
`C07K1/18 (2013.01); C07K1/32 (2013.01)
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`USPC ........................................................ .. 530/413
`(58) Field of Classi?cation Search
`CPC ............ .. C07K1/22; C07K1/18; C07K1/32;
`C07K 1/ 145
`See application ?le for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
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`5,466,377 A 11/1995 Grandics et al.
`6,660,843 B1
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`7,138,370 B2 11/2006 Oliner et a1.
`7,511,012 B2
`3/2009 Han et a1.
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`FOREIGN PATENT DOCUMENTS
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`WO97/28272
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`8/1997
`2/2009
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`OTHER PUBLICATIONS
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`Pavlinkova et a1. (Nuclear Med. Biol., 26:27-34, 1999).*
`Ronnmark et al. (J. Immunologic. Meth., 261: 199-21 1, 2002).*
`Tengliden (Development of Cleaning-In-Place Procedures for Pro
`tein a Chromatography Resins Using Design of Experiments and
`High Throughput Screening Technologies, Masters Thesis, Linkop
`ing University, Feb. 2008).*
`Amersham Biosciences (Amersham Biosciences Sepharose instruc
`tions, Edition AE, p. 1-12, 2002).*
`
`Arvidsson, P. et al., “Direct chromatographic capture of enzyme from
`crude homogenate using immobilized metal af?nity chromatography
`on a continuous supermacroporous adsorbent,” J. Chromatography
`986 (2): 276-290 (2003).
`Ling et al., “Integration of mechanical cell disruption and ?uidised
`bed recovery of G3 PHD from unclari?ed disrupted yeast: a compara
`tive study of the performance of unshielded and polymer shielded
`dye-ligand chromatography systems,” J. Biotech. 119(4): 436-448
`(2005).
`Fischer, B. et al., “Isolation renaturation and formation of disul?de
`bonds of eukaryotic proteins expressed in escherichia coli as inclu
`sion bodies,” Biotech. and Bioengineering, 41(1): 3-13 (1993).
`Ford et al., “Af?nity puri?cation of novel bispeci?c antibodies
`recognising carcinoembryonic antigen and doxorubicin,” J.
`Chromatogr. B, 754: 427-435 (2001).
`Shukla et al., “Downstream processing of monoclonal antibodiesi
`Application of platform approaches,” Journal of Chromatography B,
`848(1):28-39 (2007).
`Wang et al., “Perturbation of the antigen-binding site and
`staphylococcal protein A-binding site of IgG before signi?cant
`changes in global conformation during denaturation: an equilibrium
`study,” Biochem. J. 325(Pa1t 3):707-710 (1997).
`Hasemann & Capra, “Immunoglobulins: Structure and Function,” in
`William E. Paul, ed., Fundamental Immunology, Second Edition,
`209, 210-218 (1989).
`Ostrove, “Af?nity Chromatography: General Methods,” Guide to
`Protein Puri?cation, Methods in Enzymology 182: 371-379 (1990).
`Devereux et al., “A comprehensive set of sequence analysis programs
`for the VAX,” Nucleosides, nucleotides; & nucleic acids: Nucl. Acids
`Res. 12: 387-389 (1984).
`Gribskov and Burgess, “Sigma factors from E. coli, B. subtilis; phage
`SP01, and phage T4 are homologous proteins,” Nucl. Acids Res. 14:
`6745 (1986).
`Gulich, Susanne, et al., “Protein engineering of an IgG-binding
`domain allows milder elution conditions during af?nity chromatog
`raphy,” J. Biol. 76, Issues 2-3, pp. 233-244 (2000).
`Ostrove “Af?nity Chromatography: General Methods,” Guide to Pro
`tein Puri?cation, Methods in Enzymology 182: 357-371 (1990).
`Stoscheck, C., “Quantitation of Protein,” Guide to Protein Puri?ca
`tion, Methods in Enzymology 182: 50-68 (1990).
`Vola et al., “Recombinant proteins L and LG. Two new tools for
`puri?cation of murine antibody fragments,” Cell Biophys. 24-25:
`27-36 (1994).
`Aybay and Imir “Development of a rapid, single-step procedure using
`protein G af?nity chromatography to deplete fetal calf serum of its
`IgG and to isolate murine IgGl monoclonal antibodies from super
`natants of hybridoma cells,” Int’l. J. Immunol. Methods 233(1-2):
`77-81 (2000).
`Ejima, Daisuke, et al., Effective elution of antibodies by arginine and
`arginine derivatives in af?nity column chromatography, Analytical
`Biochem. 345 250-257 (2005).
`(Continued)
`
`Primary Examiner * Brian J Gangle
`(74) Attorney, Agent, or Firm * David B. Ran
`
`ABSTRACT
`(57)
`Methods of purifying proteins expressed in non-mammalian
`expression systems in a non-native soluble form directly from
`cell lysate are disclosed. Methods of purifying proteins
`expressed in non-mammalian expression systems in a non
`native limited solubility form directly from a refold solution
`are also disclosed. Resin regeneration methods are also pro
`vided.
`
`25 Claims, 5 Drawing Sheets
`
`Page 1
`
`KASHIV EXHIBIT 1001
`IPR2019-00791
`
`

`

`US 8,940,878 B2
`Page 2
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`
`Arakawa, Tsutomu et al., “Elution of antibodies from a Protein-A
`column by acqueous arginine solutions,” Protein Express & Purif,
`36: 244-248 (2004).
`Miller, Timothy et al., The rapid isolation of ribonuclease-free
`immuno globulin G by protein A-sepharose af?nity chromatography,
`J. Immun. Methods 24: 111-125 (1978).
`Snyder et al., “Characterization of DC-SIGN/R Interaction With
`Human Immunode?ciency Virus Type I gp I20 and ICAM Mol
`ecules favors the receptor’s role as an antigen-capturing rather than
`an adhesion receptor,” J. Virology79(8): 4589-4598, Apr. 2005.
`
`GE Healthcare Instructions 7l-7089-00AE: Af?nity media, Protein
`A Sepharose CL-4B, p. 1-8, Mar. 2006. (Cited in JP Of?ce action as
`D5 http://eclub.biomart.cn/sites/eclubbiomart.cn/themes/aktaclub/
`Files/7 l708900AEiUMiProteiniAiSepharo seiCL-4def,
`Mar. 2006).
`GE Healthcare Instructions 71-5002-60 AE: Ion exchange chroma
`tography; Q Sepharose XL, XL virus licensed, SP Sepharose XL, pp.
`1-16, Feb. 2006 (cited in JP Of?ce action as D6https://WWW.
`gelifesciences.com/gchelsiimages/GELS/Related%20Content/
`Files/13147231l6657/litdoc71500260AEi20110830185637pdf,
`Feb. 2006).
`
`* cited by examiner
`
`Page 2
`
`

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`US. Patent
`
`Jan. 27, 2015
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`Jan. 27, 2015
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`Jan.27,2015
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`US. Patent
`
`Jan. 27, 2015
`
`Sheet 5 0f 5
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`US 8,940,878 B2
`
`1
`CAPTURE PURIFICATION PROCESSES FOR
`PROTEINS EXPRESSED IN A
`NON-MAMMALIAN SYSTEM
`
`This application claims the bene?t of US. Provisional
`Application No. 61/220,477 ?led Jun. 25, 2009, which is
`incorporated by reference herein.
`
`FIELD OF THE INVENTION
`
`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.
`
`BACKGROUND OF THE INVENTION
`
`2
`matrix; (c) washing the separation matrix; and (d) eluting the
`protein from the separation matrix.
`The protein can be a complex protein, such as a protein is
`selected from the group consisting of a multimeric protein, an
`antibody and an Fc fusion protein. The non-mammalian
`expression system can comprise bacteria or yeast cells. The
`separation matrix can be an af?nity resin, such as an af?nity
`resin selected from the group consisting of Protein A, Protein
`G and a synthetic mimetic af?nity resin, or it can be a non
`af?nity resin, such as a non-af?nity resin selected from the
`group consisting of ion exchange, mixed mode, and a hydro
`phobic interaction resin. The cell lysate can be ?ltered before
`it is contacted with the separation matrix. Although not
`required, the method can further comprise refolding the pro
`tein to its native form after it is eluted from the separation
`matrix.
`A method of purifying a protein expressed in a non-native
`limited solubility form in a non-mammalian expression sys
`tem is provided. In one embodiment that method comprises
`(a) expressing a protein in a non-native limited solubility
`form in a non-mammalian cell; (b) lysing a non-mammalian
`cell; (c) solubiliZing the expressed protein in a solubilization
`solution comprising one or more of the following: (i) a dena
`turant; (ii) a reductant; and (iii) a surfactant; (d) forming a
`refold solution comprising the solubilization solution and a
`refold buffer, the refold buffer comprising one or more of the
`following: (i) a denaturant; (ii) an aggregation suppressor;
`(iii) a protein stabilizer; and (iv) a redox component; (e)
`applying the refold solution to a separation matrix under
`conditions suitable for the protein to associate with the
`matrix; (f) washing the separation matrix; and (g) eluting the
`protein from the separation matrix.
`The non-native limited solubility form can be a component
`of an inclusion body. The protein can be a complex protein,
`such as a complex protein selected from the group consisting
`of a multimeric protein, an antibody, a peptibody, and an Fc
`fusion protein. The non-mammalian expression system can
`be bacteria or yeast cells. The denaturant can comprise one or
`more of urea, guanidinium salts, dimethyl urea, methylurea
`and ethylurea, the reductant can comprise one or more of
`cysteine, DTT, beta-mercaptoethanol and glutathione, the
`surfactant can comprise one or more of sarcosyl and sodium
`dodecylsulfate, the aggregation suppressor can be selected
`from the group consisting of arginine, proline, polyethylene
`glycols, non-ionic surfactants, ionic surfactants, polyhydric
`alcohols, glycerol, sucrose, sorbitol, glucose, tris, sodium
`sulfate, potassium sulfate and osmolytes, the protein stabi
`lizer can comprise one or more of arginine, proline, polyeth
`ylene glycols, non-ionic surfactants, ionic surfactants, poly
`hydric alcohols, glycerol, sucrose, sorbitol, glucose, tris,
`sodium sulfate, potassium sulfate and osmolytes, and the
`redox component can comprise one or more of glutathione
`reduced, glutathione-oxidized, cysteine, cystine, cysteamine,
`cystamine and beta-mercaptoethanol. The separation matrix
`can be an af?nity resin such as an af?nity resin selected from
`the group consisting of Protein A, Protein G, and synthetic
`mimetic af?nity resin or the separation matrix can be a non
`af?nity resin selected from the group consisting of ion
`exchange, mixed mode, and a hydrophobic interaction resin.
`In other embodiments, the disclosed methods can further
`comprise the steps of (a) washing the separation matrix with
`a regeneration reagent; and (b) regenerating the separation
`matrix. The regeneration reagent can be one of a strong base,
`such as sodium hydroxide or a strong acid, such as phosphoric
`acid. The regenerating can comprise washing the separation
`matrix with a solution comprising one or both of a chaotrope
`present at a concentration of 4-6 M and a reductant. The
`
`Fc-containing proteins are typically expressed in mamma
`lian cells, such as CHO cells. The use of af?nity chromatog
`raphy to purify Fc-containing proteins is documented (see,
`e.g., Shukla et al., (2007) Journal 0fChr0maZography B 848
`(1)128-39) 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,
`however, are often deposited in the expressing cells in limited
`solubility forms, such as inclusion bodies, that require refold
`ing, and this has been a limiting factor in selecting non
`mammalian systems for expressing Fc-containing proteins.
`A drawback to the use of Protein A, Protein G and other
`chemistries is that in order for a protein comprising an Fc
`region to associate with the Protein A or Protein G molecule,
`the protein needs to have a minimum amount of structure.
`Often, the requisite amount of structure is absent from pro
`teins expressed recombinantly in a soluble, but non-native,
`form and consequently ProteinA chromatography is not per
`formed in a puri?cation process.
`In the case of a protein expressed in an insoluble non-native
`form, ProteinA chromatography is typically not performed in
`a puri?cation process until after the protein has been refolded
`to a degree that it can associate with the Protein A molecule
`and has been subsequently diluted out of its refold solution.
`This is because it was believed that after a protein has been
`refolded it was necessary to dilute or remove the components
`of the refold mixture in a wash step, due to the tendency of the
`components that typically make up a refold solution to disrupt
`interactions between the target protein and the Protein A
`molecules (Wang et al., (1997). Biochem. J. 325 (Part 3):707
`710). This dilution step can consume time and resources
`which, when working at a manufacturing scale of thousands
`of liters of culture, can be costly.
`The present disclosure addresses these issues by providing
`simpli?ed methods of purifying proteins comprising Fc
`regions that are expressed in non-mammalian expression sys
`tems in a non-native soluble form or in a non-native insoluble
`form.
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`SUMMARY OF THE INVENTION
`
`A method of purifying a protein expressed in a non-native
`soluble form in a non-mammalian expression system is pro
`vided. In one embodiment the method comprises (a) lysing a
`non-mammalian cell in which the protein is expressed in a
`non-native soluble form to generate a cell lysate; (b) contact
`ing the cell lysate with an separation matrix under conditions
`suitable for the protein to associate with the separation
`
`60
`
`65
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`Page 8
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`

`US 8,940,878 B2
`
`3
`chaotrope can be one of urea, dimethyl urea, methylurea,
`ethylurea, and guanidinium, and the reductant can be one of
`cysteine, DTT, beta-mercaptoethanol and glutathione. In a
`particular embodiment the regenerating comprises washing
`the separation matrix with a solution comprising 50 mM Tris,
`10 mM citrate, 6M urea, 50 mM DTT at pH 7.4.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a plot demonstrating the binding of refolded,
`non-mammalian non-native limited solubility fraction com
`plex protein, to Protein A media; in the ?gure the X denotes
`resin loading at a 9.32 min residence time, star denotes resin
`loading at a 7.68 min residence time and solid circles denote
`resin loading at a 6 min residence time.
`FIG. 2 is a table demonstrating puri?cation of a complex
`protein comprising an Fc domain using Protein A resin.
`FIG. 3 is a table demonstrating the reusability of ProteinA
`resin when used to capture a non-mammalian non-native
`limited solubility complex protein over 150 cycles using the
`disclosed methods.
`FIG. 4 is a plot demonstrating the binding pro?les of a
`refolded, non-mammalian non-native limited solubility com
`plex protein to six different ion exchange resins (IEX Resins
`1, 2, 3, 4, 5, 6, corresponding to Toyopearl SP550CTM, Toyo
`pearl SP650MTM, GigaCAP STM, POROS HSSOTM, Toyopearl
`SP650CTM and GE Healthcare SPxLTM, respectively) and a
`mixed-mode resin (MMC Resin 1, GE Healthcare MMCTM)
`following capture using the disclosed methods.
`FIG. 5 is a table demonstrating puri?cation levels achieved
`for a protein comprising an Fc domain using one anion
`exhange resin (Fractogel TMAETM) and one cation exchange
`resin (Fractogel SO3_TM).
`
`20
`
`25
`
`30
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`35
`
`The present disclosure provides methods of capturing on a
`separation matrix non-native proteins produced in microbial
`cells. In the case of the direct capture of a protein expressed in
`a non-native soluble form the advantages of the present inven
`tion over typical processes include enhanced protein concen
`tration, volume reduction, and increased recovery over tradi
`tional methods, improved protein stability, and ultimately
`process cost savings.
`In the case of the direct capture of a protein expressed in a
`non-native limited solubility form, the advantages of the
`present invention over typical processes include the elimina
`tion of the need to dilute the protein out of a refold solution
`prior to capturing it on a separation matrix.
`Another advantage of the disclosed methods is that they
`may be performed at a range of scales, from laboratory scale
`(typically milliliter or liter scale), a pilot plant scale (typically
`hundreds of liters) or on an industrial scale (typically thou
`sands of liters). The application of the disclosed methods on
`large scales may be particularly desirable, due to the potential
`savings in time and resources.
`Non-mammalian, e.g., microbial, cells can naturally pro
`duce, or can be engineered to produce, proteins that are
`expressed in either a soluble or a limited solubility form. Most
`often, engineered non-mammalian cells will deposit the
`recombinant proteins into large limited solubility aggregates
`called inclusion bodies. However, certain cell growth condi
`tions (e.g., temperature or pH) can be modi?ed to drive the
`recombinant proteins to be expressed as intracellular, soluble
`monomers. As an alternative to producing a protein of interest
`in cells in which the protein is expressed in the form of limited
`solubility inclusion bodies, cell growth conditions can be
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`modi?ed such that proteins are expressed in a non-native yet
`soluble form. The cells can then be lysed and the protein can
`be isolated by capturing it directly from cell lysate using ion
`exchange chromatography, af?nity chromatography or mixed
`mode chromatography, as described herein. The method can
`be particularly useful for purifying proteins comprising an Fc
`region.
`In one aspect, therefore, the present disclosure relates to a
`method of isolating a protein of interest comprising an Fc
`region that is expressed in a non-mammalian cell in a non
`native, yet soluble form, from a pool of lysate generated from
`the cell in which the protein was expressed. The method
`employs a separation matrix, such as Protein A. One bene?
`cial aspect of the disclosed method is that it eliminates the
`need for a refolding step before the protein is applied to the
`separation matrix. That is, non-mammalian cells expressing
`the protein of interest in a non-native soluble form can be
`lysed, the lysate applied directly to the separation matrix and
`the protein subsequently eluted from the separation matrix.
`This process allows the separation of proteins from cell cul
`tures in highly concentrated pools that can be subsequently
`refolded at high concentrations and can be of bene?t 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.
`Following isolation by the separation matrix, the protein of
`interest can optionally be subsequently refolded using any
`technique known or suspected to work well for the protein of
`interest.
`In another aspect, the present invention relates to a method
`of isolating a protein of interest comprising an Fc region that
`is expressed in a non-native limited solubility form, for
`example in inclusion bodies, that needs to be refolded and
`isolated from the refold mixture. Commonly, a refold solution
`contains a denaturant (e.g., urea or other chaotrope, organic
`solvent or strong detergent), an aggregation suppressor (e. g.,
`a mild detergent, arginine or low concentrations of PEG), a
`protein stabilizer (e.g., glycerol, sucrose or other osmolyte,
`salts) and/or a redox component (e.g., cysteine, cystine, cys
`tamine, cysteamine, glutathione). While often bene?cial for
`refolding proteins, these components can inhibit puri?cation
`(see, e.g., Wang et al., (1 997) Biochemical Journal 325 (Part
`3):707-710) and it is necessary to isolate or dilute the protein
`from these components for further processing, particularly
`before applying the protein to a separation matrix.
`In one embodiment of the disclosed method, puri?cation is
`achieved by directly applying a protein of interest, which is
`present in a refold mixture, to a separation matrix. In this
`approach, following a refold step the entire refold mixture,
`including the protein of interest, is applied directly to a sepa
`ration matrix, such as a Protein A or G resin. The protein of
`interest associates with the matrix in the presence of the
`components of refold buffer, impurities are washed away and
`the protein is eluted. Since the method omits the need for
`removing any components of the refold mixture before the
`refold mixture is applied to a separation matrix, the method
`can have the effect of saving steps, time and resources that are
`typically expended on removing the protein from refolding
`and dilution buffers in puri?cation processes. In some cases,
`the method can also reduce or eliminate the need for subse
`quent puri?cation steps.
`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 sys
`tem that have subsequently been derivatized. For example,
`following expression a protein comprising an Fc region can
`
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`US 8,940,878 B2
`
`5
`be associated with a small molecule, such as a toxin. Such
`conjugates can be puri?ed using the methods described
`herein.
`
`I. DEFINITIONS
`
`As used herein, the terms “a” and “an” mean one or more
`unless speci?cally indicated otherwise.
`As used herein, the term “non-mammalian expression sys
`tem” means a system for expressing proteins in cells derived
`from an organism other than a mammal, including but not
`limited to, prokaryotes, including bacteria such as E. coli, and
`yeast. Often a non-mammalian expression system is
`employed to express a recombinant protein of interest, while
`in other instances a protein of interest is an endogenous pro
`tein that is expressed by a non-mammalian cell. For purposes
`of the present disclosure, regardless of whether a protein of
`interest is endogenous or recombinant, if the protein is
`expressed in a non-mammalian cell then that cell is a “non
`mammalian expression system.” Similarly, a “non-mamma
`lian cell” is a cell derived from an organism other than a
`mammal, examples of which include bacteria or yeast.
`As used herein, the term “denaturant” means any com
`pound having the ability to remove some or all of a protein’s
`secondary and tertiary structure when placed in contact with
`the protein. The term denaturant refers to particular chemical
`compounds that affect denaturation, as well as solutions com
`prising a particular compound that affect denaturation.
`Examples of denaturants that can be employed in the dis
`closed method include, but are not limited to urea, guani
`dinium salts, dimethyl urea, methylurea, ethylurea and com
`binations thereof.
`As used herein, the term “aggregation suppressor” means
`any compound having the ability to disrupt and decrease or
`eliminate interactions between two or more proteins.
`Examples of aggregation suppressors can include, but are not
`limited to, amino acids such as arginine, proline, and glycine;
`polyols and sugars such as glycerol, sorbitol, sucrose, and
`trehalose; surfactants such as, polysorbate-20, CHAPS, Tri
`ton X-100, and dodecyl maltoside; and combinations thereof.
`As used herein, the term “protein stabilizer” means any
`compound having the ability to change a protein’s reaction
`equilibrium state, such that the native state of the protein is
`improved or favored. Examples of protein stabilizers can
`include, but are not limited to, sugars and polyhedric alcohols
`such as glycerol or sorbitol; polymers such as polyethylene
`glycol (PEG) and (x-cyclodextrin; amino acids salts such as
`arginine, proline, and glycine; osmolytes and certain
`Hoffmeister salts such as Tris, sodium sulfate and potassium
`sulfate; and combinations thereof.
`As used herein, the terms “Fc” and “Fc region” are used
`interchangeably and mean a fragment of an antibody that
`comprises human or non-human (e. g., murine) C H2 and C H3
`immunoglobulin domains, or which comprises two contigu
`ous regions which are at least 90% identical to human or
`non-human CH2 and CH3 immunoglobulin domains. An Fc
`can but need not have the ability to interact with an Fc recep
`tor. See, e.g., Hasemann & Capra, “Immunoglobulins: Struc
`ture and Function,” in William E. Paul, ed., Fundamental
`Immunology, Second Edition, 209, 210-218 (1989), which is
`incorporated by reference herein in its entirety.
`As used herein, the terms “protein” and “polypeptide” are
`used interchangeably and mean any chain of at least ?ve
`naturally or non-naturally occurring amino acids linked by
`peptide bonds.
`As used herein, the term “complex molecule” means any
`protein that is (a) larger than 20,000 MW, or comprises
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6
`greater than 250 amino acid residues, and (b) comprises two
`or more disul?de bonds in its native form. A complex mol
`ecule can, but need not, form multimers. Examples of com
`plex molecules include but are not limited to, antibodies,
`peptibodies and polypeptides comprising an Fc domain and
`other large proteins. Peptibodies are described in US. Pat.
`No. 6,660,843, US. Pat. No. 7,138,370 and US. Pat. No.
`7,5 1 1 ,012.
`As used herein, the term “peptibody” refers to a polypep
`tide comprising one or more bioactive peptides joined
`together, optionally via linkers, with an Fc domain. See US.
`Pat. No. 6,660,843, US. Pat. No. 7,138,370 andU.S. Pat. No.
`7,511,012 for examples of peptibodies.
`As used herein, the terms “Fc fusion” and “Fc fusion pro
`tein” are used interchangeably and refer to a peptide or
`polypeptide covalently attached to an Fc domain.
`As used herein the term “Protein A” means any protein
`identical or substantially similar to Staphylococcal ProteinA,
`including commercially available and/ or recombinant forms
`of Protein A. For the purposes of this invention, Protein A
`speci?cally 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 recom
`binant Protein A molecule, and other non-naturally occurring
`Protein A molecules.
`As used herein, the term “Protein G” means any protein
`identical or substantially similar to Streptococcal Protein G,
`including commercially available and/ or recombinant forms
`of Protein G.
`As used herein, the term “substantially similar,” when used
`in the context of a protein, including Protein A, means pro
`teins that are at least 80%, preferably at least 90% identical to
`each other in amino acid sequence and maintain or alter in a
`desirable manner the biological activity of the unaltered pro
`tein. Included in amino acids considered identical for the
`purpose of determining whether proteins are substantially
`similar are amino acids that are conservative substitutions,
`unlikely to affect biological activity, including the following:
`Ala for Ser, Val for He, Asp for Glu, Thr for Ser, Ala for Gly,
`Ala for Thr, Ser for Asn, Ala for Val, Ser for Gly, Tyr for Phe,
`Ala for Pro, Lys forArg, Asp forAsn, Leu for He, Leu for Val,
`Ala for Glu, Asp for Gly, and these changes in the reverse.
`See, e.g., Neurath et al., The Proteins, Academic Press, New
`York (1979). The percent identity of two amino sequences
`can be determined by visual inspection and mathematical
`calculation, or more preferably, the comparison is done by
`comparing sequence information using a computer program
`such as the Genetics Computer Group (GCG; Madison, Wis.)
`Wisconsin package version 10.0 program, “GAP” (Devereux
`et al., 1984, Nucl. Acids Res. 12: 387) or other comparable
`computer programs. The preferred default parameters for the
`“GAP” program includes: (1) the weighted amino acid com
`parison matrix of Gribskov and Burgess ((1986), Nucl. Acids
`Res. 14: 6745), as described by SchwartZ and Dayhoff, eds.,
`Atlas of Polypeptide Sequence and Structure, National Bio
`medical Research Foundation, pp. 353-358 (1979), or other
`comparable comparison matrices; (2) a penalty of 30 for each
`gap and an additional penalty of 1 for each symbol in each gap
`for amino acid sequences; (3) no penalty for end gaps; and (4)
`no maximum penalty for long gaps. Other programs used by
`those skilled in the art of sequence comparison can also be
`used.
`As used herein, the terms “isolate” and “purify” are used
`interchangeably and mean to reduce by 1%, 2%, 3%, 4%, 5%,
`10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
`60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or more, the
`
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`US 8,940,878 B2
`
`7
`amount of heterogenous elements, for example biological
`macromolecules such as proteins or DNA, that may be
`present in a sample comprising a protein of interest. The
`presence of heterogenous proteins can be assayed by any
`appropriate method including High-performance Liquid
`Chromatography (HPLC), gel electrophoresis and staining
`and/ or ELISA assay. The presence of DNA and other nucleic
`acids can be assayed by any appropriate method including gel
`electrophoresis and staining and/or assays employing poly
`merase chain reaction.
`As used herein, the term “separation matrix” means any
`adsorbent material that utilizes speci?c, reversible interac
`tions between synthetic and/or biomolecules, e.g., the prop
`erty of ProteinA to bind to an Fc region of an IgG antibody or
`other Fc-containing protein, in order to effect the separation
`of the protein from its environment. In other embodiments the
`speci?c, reversible interactions can be base on a property such
`as isoelectric point, hydrophobicity, or size. In one particular
`embodiment, a separation matrix comprises an adsorbent,
`such as ProteinA, af?xed to a solid support. See, e.g., Ostrove
`(1990) in “Guide to Protein Puri?cation,” Methods in Enzy
`mology 182: 357-379, which is incorporated herein in its
`entirety.
`As used herein, the terms “non-native” and “non-native
`form” are used interchangeably and when used in the context
`of a protein of interest, such as a protein comprising a Fc
`domain, mean that the protein lacks at least one formed struc
`ture attribute found in a form of the protein that is biologically
`active in an appropriate in vivo or in vitro assay designed to
`assess the protein’s biological activity. Examples of structural
`features that can be lacking in a non-native form of a protein
`can include, but are not limited to, a disul?de bond, quater
`nary structure, disrupted secondary or tertiary structure or a
`state that makes the protein biologically inactive in an appro
`priate assay. A protein in a non-native form can but need not
`form aggregates.
`As used herein, the term “non-native soluble form” when
`used in the context of a protein of interest, such as a pr