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`A-1441-US-DIV
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`Attorney Docket No.
`
`First Named Inventor
`
`Joseph SHULTZ
`
`Title
`
`Capture PurI1IcatIon Processes 1or Proteins Expressed In a Non-Mammalian System
`
`UTILITY
`PATENT APPLICATION
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`Date
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`January 16, 2015
`Registration No. 61,000
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`(Attorney/Agent)
`
`1 of 196
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`Fresenius Kabi
`Exhibit 1033
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`

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`2 of 196
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`CAPTURE PURIFICATION PROCESSES FOR PROTEINS EXPRESSED IN A NON(cid:173)
`
`MAMMALIAN SYSTEM
`
`This application claims the benefit of U.S. Provisional Application No.
`
`61/220,477 filed June 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
`
`Fe-containing proteins are typically expressed in mammalian cells, such as CHO
`
`cells. The use of affinity chromatography to purify Fe-containing proteins is documented
`
`(see, e.g., Shukla et al., (2007) Journal of Chromatography B 848(1):28-39) and is
`
`successful, in part, due to the degree of Fe structure observed in proteins expressed in
`
`such systems. Fe-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 refolding, and this has been a limiting factor in selecting non(cid:173)
`
`mammalian systems for expressing Fe-containing proteins.
`
`A drawback to the use of Protein A, Protein G and other chemistries is that in
`
`order for a protein comprising an Fe 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 proteins expressed recombinantly in a soluble, but
`
`non-native, form and consequently Protein A chromatography is not performed in a
`
`purification process.
`
`In the case of a protein expressed in an insoluble non-native form, Protein A
`
`chromatography is typically not performed in a purification 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
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`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 simplified methods of
`
`purifying proteins comprising Fe regions that are expressed in non-mammalian
`
`expression systems in a non-native soluble form or in a non-native insoluble form.
`
`SUMMARY OF THE INVENTION
`
`A method of purifying a protein expressed in a non-native soluble form in a non(cid:173)
`
`mammalian expression system is provided. 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) contacting the cell lysate with an separation matrix
`
`under conditions suitable for the protein to associate with the separation 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 Fe fusion protein. The non(cid:173)
`
`mammalian expression system can comprise bacteria or yeast cells. The separation
`
`matrix can be an affinity resin, such as an affinity resin selected from the group
`
`consisting of Protein A, Protein G and a synthetic mimetic affinity resin, or it can be a
`
`non-affinity resin, such as a non-affinity resin selected from the group consisting of ion
`
`exchange, mixed mode, and a hydrophobic interaction resin. The cell lysate can be
`
`filtered before it is contacted with the separation matrix. Although not required, the
`
`method can further comprise refolding the protein 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 system is provided. In one embodiment that method
`
`comprises (a) expressing a protein in a non-native limited solubility form in a non(cid:173)
`
`mammalian cell; (b) lysing a non-mammalian cell; ( c) solubilizing the expressed protein
`
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`in a solubilization solution comprising one or more of the following: (i) a denaturant; (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 Fe 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
`
`ethyl urea,
`
`the reductant can comprise one or more of cysteine, DTT, beta(cid:173)
`
`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 stabilizer can comprise one or more of
`
`arginine, proline, polyethylene glycols, non-ionic surfactants,
`
`ionic surfactants,
`
`polyhydric 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 affinity resin such as an affinity
`
`resin selected from the group consisting of Protein A, Protein G, and synthetic mimetic
`
`affinity resin or the separation matrix can be a non-affinity 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
`
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`present at a concentration of 4-6 M and a reductant. The 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 50mM
`
`Tris, lOmM citrate, 6M urea, 50mM DTT at pH 7.4.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Figure 1 is a plot demonstrating the binding of refolded, non-mammalian non(cid:173)
`
`native limited solubility fraction complex protein, to Protein A media; in the figure 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.
`
`Figure 2 is a table demonstrating purification of a complex protein comprising an
`
`F c domain using Protein A resin.
`
`Figure 3 is a table demonstrating the reusability of Protein A resin when used to
`
`capture a non-mammalian non-native limited solubility complex protein over 150 cycles
`
`using the disclosed methods.
`
`Figure 4 is a plot demonstrating the binding profiles of a refolded, non(cid:173)
`
`mammalian non-native limited solubility complex protein to six different ion exchange
`
`resins (IEX Resins 1, 2, 3, 4, 5, 6, corresponding to Toyopearl SP550C™, Toyopearl
`
`SP650M™, GigaCAP S™, POROS HS50™, Toyopearl SP650C™ and GE Healthcare
`
`SPxL™, respectively) and a mixed-mode resin (MMC Resin 1, GE Healthcare MMC™)
`
`following capture using the disclosed methods.
`
`Figure 5 is a table demonstrating purification levels achieved for a protein
`
`comprising an Fe domain using one anion exhange resin (Fractogel TMAE™) and one
`
`cation exchange resin (Fractogel SO3-™).
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The present disclosure provides methods of capturing on a separation matrix non(cid:173)
`
`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 invention over
`
`typical processes include enhanced protein concentration, volume reduction, and
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`increased recovery over traditional 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
`
`elimination 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 thousands 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 produce, 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 conditions (e.g., temperature or pH) can be modified 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 modified
`
`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, affinity chromatography or mixed mode chromatography, as
`
`described herein. The method can be particularly useful for purifying proteins
`
`comprising an F c region.
`
`In one aspect, therefore, the present disclosure relates to a method of isolating a
`
`protein of interest comprising an Fe 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
`
`beneficial 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
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`applied directly to the separation matrix and the protein subsequently eluted from the
`
`separation matrix. 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.
`
`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 Fe 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,
`
`cystamine, cysteamine, glutathione ). While often beneficial for refolding proteins, these
`
`components can inhibit purification (see, e. g., Wang et al., (1997) 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, purification 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 separation 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
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`buffers in purification processes. In some cases, the method can also reduce or eliminate
`
`the need for subsequent purification 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 system that have subsequently been derivatized. For example, following
`
`expression a protein comprising an Fe region can be associated with a small molecule,
`
`such as a toxin. Such conjugates can be purified using the methods described herein.
`
`As used herein, the terms "a" and "an" mean one or more unless specifically
`
`I.
`
`Definitions
`
`indicated otherwise.
`
`As used herein, the term "non-mammalian expression system" 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(cid:173)
`
`mammalian expression system is employed to express a recombinant protein of interest,
`
`while in other instances a protein of interest is an endogenous protein 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(cid:173)
`
`mammalian cell then that cell is a "non-mammalian expression system." Similarly, a
`
`"non-mammalian 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 compound 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 comprising a particular compound that affect
`
`denaturation. Examples of denaturants that can be employed in the disclosed method
`
`include, but are not limited to urea, guanidinium salts, dimethyl urea, methylurea,
`
`ethylurea and combinations 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
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`such as arginine, proline, and glycine; polyols and sugars such as glycerol, sorbitol,
`
`sucrose, and trehalose; surfactants such as, polysorbate-20, CHAPS, Triton 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 a-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 "Fe" and "Fe region" are used interchangeably and
`
`mean a fragment of an antibody that comprises human or non-human (e.g., murine) Ctt2
`
`and Cm immunoglobulin domains, or which comprises two contiguous regions which are
`
`at least 90% identical to human or non-human Ctt2 and Cm immunoglobulin domains.
`
`An Fe can but need not have the ability to interact with an Fe receptor. See, e.g.,
`
`Hasemann & Capra, "Immunoglobulins: Structure 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 five 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 greater than 250 amino acid residues, and (b) comprises
`
`two or more disulfide bonds in its native form. A complex molecule can, but need not,
`
`form multimers. Examples of complex molecules include but are not limited to,
`
`antibodies, peptibodies and polypeptides comprising an Fe domain and other large
`
`proteins. Peptibodies are described in US Patent No 6,660,843, US Patent No 7,138,370
`
`and US Patent No 7,511,012.
`
`As used herein, the term "peptibody" refers to a polypeptide comprising one or
`
`more bioactive peptides joined together, optionally via linkers, with an Fe domain. See
`
`8
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`10 of 196
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`Fresenius Kabi
`Exhibit 1033
`
`

`

`US Patent No 6,660,843, US Patent No 7,138,370 and US Patent No 7,511,012 for
`
`examples of peptibodies.
`
`As used herein, the terms "F c fusion" and "F c fusion protein" are used
`
`interchangeably and refer to a peptide or polypeptide covalently attached to an Fe
`
`domain.
`
`As used herein the term "Protein A" means any protein identical or substantially
`
`similar
`
`to Staphylococcal Protein A,
`
`including commercially available and/or
`
`recombinant forms of Protein A. For the purposes of this invention, Protein A
`
`specifically includes engineered Protein A derived media, such as Mab Select SuRe™
`
`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.
`
`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 proteins 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 protein.
`
`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 Ile, 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 for Arg,
`
`Asp for Asn, Leu for Ile, 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
`
`9
`
`11 of 196
`
`Fresenius Kabi
`Exhibit 1033
`
`

`

`acid comparison 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 Biomedical 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 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(cid:173)
`
`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
`
`polymerase chain reaction.
`
`As used herein, the term "separation matrix" means any adsorbent material that
`
`utilizes specific, reversible interactions between synthetic and/or biomolecules, e.g., the
`
`property of Protein A to bind to an Fe region of an IgG antibody or other Fe-containing
`
`protein, in order to effect the separation of the protein from its environment. In other
`
`embodiments the specific, 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 Protein A, affixed to a solid support. See, e.g.,
`
`Ostrove (1990) in "Guide to Protein Purification," Methods in Enzymology 182: 357-379,
`
`