1111111111111111 IIIIII IIIII 11111 1111111111 lllll 111111111111111 1111111111 1111111111 11111111
`US 20060172384Al
`
`c19) United States
`c12) Patent Application Publication
`Reardon et al.
`
`c10) Pub. No.: US 2006/0172384 Al
`Aug. 3, 2006
`(43) Pub. Date:
`
`(54) FGF18 PRODUCTION IN PROKARYOTIC
`HOSTS
`
`(76)
`
`Inventors: Brian J. Reardon, Seattle, WA (US);
`Susan H. Julien, Seattle, WA (US);
`Chung-Ieung Chan, Sammamish, WA
`(US); Hong Y. Liu, Seattle, WA (US)
`
`Correspondence Address:
`ZymoGenetics, Inc.
`1201 Eastlake Avenue East
`Seattle, WA 98102 (US)
`
`(21) Appl. No.:
`
`11/301,383
`
`(22) Filed:
`
`Dec. 12, 2005
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/635,023, filed on Dec.
`10, 2004.
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`C12P 21106
`(2006.01)
`C07H 21104
`(2006.01)
`C12N 15174
`(2006.01)
`C12N 1121
`(2006.01)
`C07K 14150
`(2006.01)
`(52) U.S. Cl. ................... 435/69.1; 435/252.33; 435/488;
`530/399; 536/23.5
`
`(57)
`
`ABSTRACT
`
`The expression vectors and methods using an E. coli expres(cid:173)
`sion system for the large scale production of FGF18 are
`described. The vectors utilize the FGF18 coding sequence
`with specific changes in nucleotides in order to optimize
`codons and mRNA secondary structure for translation in E.
`coli. Using the expression vectors, the FGF18 gene was
`produced in E. coli to a level of greater than 1 g/L in fed
`batch fermentation. Also included are OmpT deficient E.
`coli strains, as well as OmpT and fhuA negative strains
`transformed with an FGF18 expression vector.
`
`1 of 48
`
`Fresenius Kabi
`Exhibit 1004
`
`

`

`Patent Application Publication Aug. 3, 2006 Sheet 1 of 2
`
`US 2006/0172384 Al
`
`ColEl
`
`pSDH170_ 1 - yeast
`(5262 bp)
`
`-10 -35
`RBS 4\
`
`Notl
`
`FIG. 1
`
`2 of 48
`
`Fresenius Kabi
`Exhibit 1004
`
`

`

`Patent Application Publication Aug. 3, 2006 Sheet 2 of 2
`
`US 2006/0172384 Al
`
`Pvull
`
`pSDH17 4_ 1 - yeast
`(5230 bp)
`
`Pvul
`
`Notl
`
`FIG. 2
`
`3 of 48
`
`Fresenius Kabi
`Exhibit 1004
`
`

`

`US 2006/0172384 Al
`
`Aug. 3, 2006
`
`1
`
`FGF18 PRODUCTION IN PROKARYOTIC HOSTS
`
`[0001] This application claims the benefit of U.S. Provi(cid:173)
`sional Application Ser. No. 60/635,023, filed Dec. 10, 2004,
`which is herein incorporated by reference.
`
`BACKGROUND OF THE INVENTION
`
`[0002] The increased availability and identification of
`genes from human and other genomes has led to an
`increased need for efficient expression and purification of
`recombinant proteins. The expression of proteins in bacteria
`is by far the most widely used approach for the production
`of cloned genes. For many reasons, expression in bacteria is
`preferred to expression in eukaryotic cells. For example,
`bacteria are much easier to grow than eukaryotic cells. More
`specifically, the availability of a wealth of sophisticated
`molecular genetic tools and thousands of mutants make E.
`coli, as an expression host, extremely useful for protein
`production. However, the high-level production of func(cid:173)
`tional proteins in E. coli., especially those from eukaryotic
`sources has often been difficult.
`
`[0003] FGF18 is a member of the fibroblast growth factor
`family that shares significant sequence homology with
`FGF8 and FGF17. These three factors are thought to com(cid:173)
`prise a subfamily of the FGF proteins. As with all members
`of the FGF family, FGF18 has discrete effects on both
`developing and adult tissues and is thought to play a part in
`embroyonic development and wound healing (see, e.g.,
`Ornitz and Marie, Genes & Development, 16:1446-1465
`(discussing the role of all FGFs, including FGF18 in bone
`development)). In particular, FGF18 has been shown to have
`proliferative effects on cartilage and neural tissues, among
`others (Ellsworth et al., Osteoarthritis and Cartilage (2002)
`10, 308-320; Ellsworth et al. Stroke (2003) 34(6): 1507-12).
`
`[0004] Recombinant FGF18 has been produced
`in
`prokaryotic cells, in particular E. coli. The resulting bacterial
`produced protein is not glycosylated and is produced in an
`aggregated state. Initial experiments indicated that FGF18
`produced in bacterial resulted in truncation of the protein,
`producing truncated FGF18 (trFGF18). As the truncated
`version appeared to have very similar, if not identical,
`biological properties as the full-length protein, constructs
`were then made that produced solely the truncated version.
`Production of FGF 18 or trFGF 18 from E. coli requires that
`the aggregated proteins be solubilized from the insoluble
`inclusion bodies and renatured or refolded. Without rena(cid:173)
`turation, the specific activity of the recombinant protein will
`be significantly reduced.
`
`[0005] Despite advances in the expression ofrecombinant
`proteins in bacterial hosts, there exists a need for improved
`methods for producing biologically active and purified
`recombinant FGF18 and trFGF18 proteins in prokaryotic
`systems that result in higher yields for protein production.
`These and other aspects of the invention will become
`evident upon reference to the following detailed description.
`In addition, various references are identified below and are
`incorporated by reference in their entirety.
`
`SUMMARY OF THE INVENTION
`
`[0006]
`In one aspect, the present invention provides an
`expression vector for producing FGFl 8 or trFGFl 8 proteins
`comprising the operably linked elements of a prokaryotic
`
`origin of replication, a transcriptional initiation DNA ele(cid:173)
`ment, and polynucleotide sequence and a transcriptional
`terminator. In another aspect, the expression vector is the
`vectorpSDH170 (SEQ ID NO:1) that can be used to produce
`FGF18. In another aspect, the expression vector is pSDHl 74
`(SEQ ID NO:2) that can be used to produce trFGF18.
`Further embodiments provide the expression vector can
`include a selectable marker.
`
`[0007]
`In another aspect, the present invention provides
`prokaryotic host cells transformed the expression vectors
`described as comprising SEQ ID NO: 3:, a polynucleotide
`sequence encoding the polypeptide of SEQ ID NO:4, or
`vector pSDHl 70 (SEQ ID NO: 1 ). In another aspect, the
`present invention provides prokaryotic host cells trans(cid:173)
`formed the expression vectors described as comprising SEQ
`ID NO:5, a polynucleotide sequence encoding the polypep(cid:173)
`tide of SEQ ID NO:6, or vector pSDHl 74 (SEQ ID NO:2).
`In other embodiments, the host strain is E. coli strain W3110
`or the strain zGOLDl or zGOLD5.
`
`[0008]
`In another aspect, the present invention provides
`methods for producing FGF18 or trFGF18 proteins under
`conditions wherein the FGF18 or trFGF18 protein is
`expressed. In one embodiment, the method comprises cul(cid:173)
`turing a host cell expressing FGF18 after being transformed
`with pSDHl 70. In a second embodiment, the method com(cid:173)
`prises culturing a host cell expressing trFGF18 after being
`transformed with pSDHl 74. In other embodiments, the
`method comprising culturing a host cell transformed with an
`expression vector comprising SEQ ID NO:1. In further
`embodiments, the method comprising culturing a host cell
`transformed with an expression vector comprising SEQ ID
`NO:2. The method also comprises recovering the host cells
`from the growth medium, and then isolating the FGF18 or
`trFGF18 protein from the host cells.
`
`[0009]
`In other aspects, the present invention provides
`methods for producing FGF18 or trFGF18 comprising the
`steps as described above, in a fed batch fermentation process
`or a batch fermentation process.
`
`[0010]
`In another aspect, the present invention provides
`methods for producing an FGF18 or trFG18 protein com(cid:173)
`prising culturing a host cell as described above in a shake
`flask to an OD600 of 5 to 20 in a growth medium, inoculating
`a fermentation vessel with 1 to 12% volume to volume (v/v)
`of shake flask medium containing host cells, culturing the
`host cells in a growth medium at a pH of 6.2 to 7.2, where
`a feed solution is fed into the fermentation vessel before 15
`hours elapsed fermentation time (EFT), adding an inducing
`agent to the fermentation vessel at 20 to 30 hours EFT, and
`harvesting the host cells at 48 to 56 hours EFT. In one
`embodiment, the inducing agent is isopropyl ~-D thiogalac(cid:173)
`topyranoside (IPTG) at 0.5 to 2 mM. In another embodi(cid:173)
`ment, the feed solution comprises a carbohydrate selected
`from the group consisting of glycerol and glucose and the
`feed is 5 to 15 grams of carbohydrate per hour. In another
`embodiment, the glycerol in the feed solution is 40 to 70%
`v/v glycerol or the glucose is 40 to 70% w/v glucose. In
`further embodiments, the glycerol is about 70% v/v or the
`glucose is about 60% w/v.
`
`[0011]
`In one aspect, the present invention provides meth(cid:173)
`ods of producing FGF 18 or trFGF 18 comprising seeding a
`flask with an inoculum comprising an E. coli W3110 host
`cells expressing an FGF 18 or trFGF 18 polypeptide as shown
`
`4 of 48
`
`Fresenius Kabi
`Exhibit 1004
`
`

`

`US 2006/0172384 Al
`
`Aug. 3, 2006
`
`2
`
`in SEQ ID NO:4 or SEQ ID NO: 6, or an E. coli W3110 host
`cell comprising pSDHl 70 or pSDHl 74 vector, wherein an
`FGF18 or trFGF18 polypeptide is expressed, and with
`growth medium comprising about 5 g/1 glycerol, culturing
`the inoculum in a growth medium for 16 to 20 hours at about
`30° C., transferring the cultured inoculum in growth medium
`to a batch fermentator at a concentration 0.5 to 5% v/v
`inoculum, fermenting the batch fermentation at about 37° C.
`and about pH 6.8 with about 2% glycerol, introducing a
`glucose feed at about 8 hours EFT of about 9.5 g glucose/
`liter/hour and continuing until end of a fermentation run,
`adding IPTG at about 24 hours EFT to final concentration of
`0.5 to 2 mM, fermenting about 28 hours ofIPTG, harvesting
`fermentation broth from the fermentor, adding an equal
`volume of water to the fermentation broth, and homogeniz(cid:173)
`ing and centrifuging to collect a cell pellet or cell slurry
`comprising FGF18 or trFGF18 protein material.
`
`[0012]
`In another aspect, the present invention provides
`methods for isolating insoluble FGF18 or trFGF18 protein
`comprising a sequence of amino acid residues as shown in
`SEQ ID NO:4 or SEQ ID NO:6 comprising separating water
`insoluble FGF18 or trFGF18 protein from a cell pellet or
`slurry, dissolving the insoluble FGF18 or trFGF18 material
`in a chaotropic solvent, diluting the chaotropic solvent and
`refolding the FGF18 or trFGF18 protein; and isolating the
`FGF18 or trFGF18 protein, wherein the isolated protein is
`capable of being biologically active. In one embodiment of
`the invention, the isolated FGF18 or trFGF18 protein is at
`least 90% pure. In another embodiment, the isolated FGF18
`or trFGF 18 protein is at least 90% pure and has an endotoxin
`level of less that 10 endotoxin units per mg FGF18 or
`trFGF18 protein.
`
`[0013]
`In another aspect, the present invention provides
`methods for isolating insoluble FGF18 or trFGF18 protein
`comprising a sequence of amino acid residues as shown in
`SEQ ID NO:4 or SEQ ID NO:6 comprising separating from
`a fermentation broth a cell pellet or cell slurry comprising
`water insoluble FGFl 8 or trFGFl 8 protein material, homog(cid:173)
`enizing the cell pellet or cell slurry to collect inclusion
`bodies, dissolving the insoluble FGF18 or trFGF18 protein
`material in a chaoptropic solvent comprising a guanidine
`salt, diluting the chaotropic solvent by addition of a refold(cid:173)
`ing buffer, isolating the FGF18 or trFGF18 protein by
`removing unfolded and aggregated proteins by filtering, and
`purifying the FGF18 or trFGF18 refolded protein on a cation
`exchange colunm, wherein the isolated and purified FGF18
`or trFGF 18 protein is capable of being biologically active.
`
`[0014]
`In another aspect, the present invention provides a
`method for isolating insoluble FGF18 or trFGF18 protein
`comprising a sequence of amino acid residues as shown in
`SEQ ID NO:4 or SEQ ID NO:6 comprising separating from
`a fermentation broth a cell pellet or cell slurry comprising
`water insoluble FGF18 or trFGF18 material, homogenizing
`the cell pellet or cell slurry to collect inclusion bodies,
`dissolving the insoluble FGF 18 or trFGF 18 protein material
`in a chaotropic solvent comprising a guanidine salt, diluting
`the chaotropic solvent by addition of a refolding buffer,
`isolating the FGF18 or trFGF18 protein by removing
`unfolded and aggregated proteins by filtering, purifying the
`FGF18 or trFGF18 refolded protein on a cation exchange
`colunm, and purifying the FGF18 or trFGF18 eluate on a
`
`hydrophobic interaction colunm, wherein the isolated and
`purified FGF18 or trFGF18 protein is capable of being
`biologically active.
`
`[0015]
`In other embodiments, the above methods for iso(cid:173)
`lating insoluble FGF18 or trFGF18 protein comprise mea(cid:173)
`suring biological activity using an FGF18 receptor binding
`assays.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`[0016] FIG. 1 is
`illustration of expression plasmid
`pSDHl 70, which comprises the codon optimized nucleotide
`sequence for FGF18.
`
`[0017] FIG. 2
`illustration of expression plasmid
`is
`pSDHl 74, which comprises the codon optimized nucleotide
`sequence for trFGF18.
`
`DESCRIPTION OF THE INVENTION
`
`[0018] The following definitions are provided to facilitate
`understanding of the invention.
`[0019] As used herein, "nucleic acid" or "nucleic acid
`molecule" refers to polynucleotides, such as deoxyribo(cid:173)
`nucleic acid (DNA) or ribonucleic acid (RNA), oligonucle(cid:173)
`otides, fragments generated by the polymerase chain reac(cid:173)
`tion (PCR), and fragments generated by any of ligation,
`scission, endonuclease action, and exonuclease action.
`Nucleic acid molecules can be composed of monomers that
`are naturally-occurring nucleotides (such as DNA and
`RNA), or analogs of naturally-occurring nucleotides (e.g.,
`a-enantiomeric forms of naturally-occurring nucleotides),
`or a combination of both. Modified nucleotides can have
`alterations in sugar moieties and/or in pyrimidine or purine
`base moieties. Sugar modifications include, for example,
`replacement of one or more hydroxyl groups with halogens,
`alkyl groups, amines, and azido groups, or sugars can be
`functionalized as ethers or esters. Moreover, the entire sugar
`moiety can be replaced with sterically and electronically
`similar structures, such as aza-sugars and carbocyclic sugar
`analogs. Examples of modifications in a base moiety include
`alkylated purines and pyrimidines, acylated purines or pyri(cid:173)
`midines, or other well-known heterocyclic substitutes.
`Nucleic acid monomers can be linked by phosphodiester
`bonds or analogs of such linkages. Analogs of phosphodi(cid:173)
`ester linkages include phosphorothioate, phosphorodithio(cid:173)
`ate, phosphoroselenoate, phosphorodiselenoate, phospho(cid:173)
`roanilothioate, phosphoranilidate, phosphoramidate, and the
`like. The term "nucleic acid molecule" also includes so(cid:173)
`called "peptide nucleic acids," which comprise naturally(cid:173)
`occurring or modified nucleic acid bases attached to a
`polyamide backbone. Nucleic acids can be either single
`stranded or double stranded.
`
`[0020] The term "complement of a nucleic acid molecule"
`refers to a nucleic acid molecule having a complementary
`nucleotide sequence and reverse orientation as compared to
`a reference nucleotide sequence.
`
`[0021] An "enhancer" is a type ofregulatory element that
`can increase the efficiency of transcription, regardless of the
`distance or orientation of the enhancer relative to the start
`site of transcription.
`
`[0022]
`"Heterologous DNA" refers to a DNA molecule, or
`a population ofDNAmolecules, that does not exist naturally
`
`5 of 48
`
`Fresenius Kabi
`Exhibit 1004
`
`

`

`US 2006/0172384 Al
`
`Aug. 3, 2006
`
`3
`
`within a given host cell. DNA molecules heterologous to a
`particular host cell may contain DNA derived from the host
`cell species (i.e., endogenous DNA) so long as that host
`DNA is combined with non-host DNA (i.e., exogenous
`DNA). For example, a DNA molecule containing a non-host
`DNA segment encoding a polypeptide operably linked to a
`host DNA segment comprising a transcription promoter is
`considered to be a heterologous DNA molecule. Conversely,
`a heterologous DNA molecule can comprise an endogenous
`gene operably linked with an exogenous promoter. As
`another illustration, a DNA molecule comprising a gene
`derived from a wild-type cell is considered to be heterolo(cid:173)
`gous DNA if that DNA molecule is introduced into a mutant
`cell that lacks the wild-type gene.
`[0023] The term "contig" denotes a nucleic acid molecule
`that has a contiguous stretch of identical or complementary
`sequence to another nucleic acid molecule. Contiguous
`sequences are said to "overlap" a given stretch of a nucleic
`acid molecule either in their entirety or along a partial stretch
`of the nucleic acid molecule.
`
`[0024]
`"Complementary DNA (cDNA)" is a single(cid:173)
`stranded DNA molecule that is formed from an mRNA
`template by the enzyme reverse transcriptase. Typically, a
`primer complementary to portions of mRNA is employed for
`the initiation of reverse transcription. Those skilled in the art
`also use the term "cDNA" to refer to a double-stranded DNA
`molecule consisting of such a single-stranded DNA mol(cid:173)
`ecule and its complementary DNA strand. The term "cDNA"
`also refers to a clone of a cDNA molecule synthesized from
`an RNA template.
`[0025] An "isolated nucleic acid molecule" is a nucleic
`acid molecule that is not integrated in the genomic DNA of
`an organism. For example, a DNA molecule that encodes a
`growth factor that has been separated from the genomic
`DNA of a cell is an isolated DNA molecule. Another
`example of an isolated nucleic acid molecule is a chemi(cid:173)
`cally-synthesized nucleic acid molecule that is not integrated
`in the genome of an organism. A nucleic acid molecule that
`has been isolated from a particular species is smaller than the
`complete DNA molecule of a chromosome from that spe(cid:173)
`cies.
`
`[0026]
`"Linear DNA" denotes non-circular DNA mol(cid:173)
`ecules with free 5' and 3' ends. Linear DNA can be prepared
`from closed circular DNA molecules, such as plasmids, by
`enzymatic digestion or physical disruption.
`
`[0027] A "promoter" is a nucleotide sequence that directs
`the transcription of a structural gene. Typically, a promoter
`is located in the 5' non-coding region of a gene, proximal to
`the transcriptional start site of a structural gene. Sequence
`elements within promoters that function in the initiation of
`transcription are often characterized by consensus nucle(cid:173)
`otide sequences. These promoters include, for example, but
`are not limited to, IPTG-inducible promoters, bacteriophage
`T7 promoters and bacteriophage "-PL· See Sambrook et al.,
`Molecular Cloning: A Laboratory Manual, 3rd ed., Cold
`Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
`2001. A typical promoter will have three components, con(cid:173)
`sisting of consensus sequences at -35 and -10 with a
`sequence of between 16 and 19 nucleotides between them
`(Lisset, S. and Margalit, H., Nucleic Acids Res. 21: 1512,
`1993). Promoters of this sort include the lac, trp, trp-lac (tac)
`and trp-lac(trc) promoters. If a promoter is an inducible
`
`promoter, then the rate of transcription increases in response
`to an inducing agent. In contrast, the rate of transcription is
`not regulated by an inducing agent if the promoter is a
`constitutive promoter. Repressible promoters are also
`known.
`[0028] A "core promoter" contains essential nucleotide
`sequences for promoter function, including the start of
`transcription. By this definition, a core promoter may or may
`not have detectable activity in the absence of specific
`sequences that may enhance the activity or confer tissue
`specific activity.
`[0029] A "regulatory element" is a nucleotide sequence
`that modulates the activity of a core promoter. For example,
`a eukaryotic regulatory element may contain a nucleotide
`sequence that binds with cellular factors enabling transcrip(cid:173)
`tion exclusively or preferentially in particular cells, tissues,
`or organelles. These types of regulatory elements are nor(cid:173)
`mally associated with genes that are expressed in a "cell(cid:173)
`specific,""tissue-specific," or "organelle-specific" mamier.
`Bacterial promoters have regulatory elements that bind and
`modulate the activity of the core promoter, such as operator
`sequences that bind activator or repressor molecules.
`[0030] A "cloning vector" is a nucleic acid molecule, such
`as a plasmid, cosmid, or, bacteriophage, which has the
`capability of replicating autonomously in a host cell. Clon(cid:173)
`ing vectors typically contain one or a small number of
`restriction endonuclease recognition sites that allow inser(cid:173)
`tion of a nucleic acid molecule in a determinable fashion
`without loss of an essential biological function of the vector,
`as well as nucleotide sequences encoding a marker gene that
`is suitable for use in the identification and selection of cells
`transformed with the cloning vector. Marker genes typically
`include genes that provide resistance to antibiotic.
`[0031] An "expression vector" is a nucleic acid molecule
`encoding a gene that is expressed in a host cell. Typically, an
`expression vector comprises a transcriptional promoter, a
`gene, an origin of replication, a selectable marker, and a
`transcriptional terminator. Gene expression is usually placed
`under the control of a promoter, and such a gene is said to
`be "operably linked to" the promoter. Similarly, a regulatory
`element and a core promoter are operably linked if the
`regulatory element modulates the activity of the core pro(cid:173)
`moter. An expression vector may also be known as an
`expression construct.
`[0032] A "recombinant host" is a cell that contains a
`heterologous nucleic acid molecule, such as a cloning vector
`or expression vector.
`[0033] The term "expression" refers to the biosynthesis of
`a gene product. For example, in the case of a structural gene,
`expression involves transcription of the structural gene into
`mRNA and the translation of mRNA into one or more
`polypeptides.
`[0034] The term "secretory signal sequence" denotes a
`DNA sequence that encodes a peptide (a "secretory pep(cid:173)
`tide") that, as a component of a larger polypeptide, directs
`the larger polypeptide through a secretory pathway of a cell
`in which it is synthesized. The larger polypeptide is com(cid:173)
`monly cleaved to remove the secretory peptide during transit
`through the secretory pathway.
`[0035] A "polypeptide" is a polymer of amino acid resi(cid:173)
`dues joined by peptide bonds, whether produced naturally or
`
`6 of 48
`
`Fresenius Kabi
`Exhibit 1004
`
`

`

`US 2006/0172384 Al
`
`Aug. 3, 2006
`
`4
`
`synthetically. Polypeptides of less than about 10 amino acid
`residues are commonly referred to as "peptides."
`
`[0036] A "protein" is a macromolecule comprising one or
`more polypeptide chains. A protein may also comprise
`non-peptidic components, such as carbohydrate groups. Car(cid:173)
`bohydrates and other non-peptidic substituents may be
`added to a protein. Proteins are defined herein in terms of
`their amino acid backbone structures; substituents such as
`carbohydrate groups and non-peptidic groups are generally
`not specified, but may be present nonetheless.
`
`[0037] A peptide or polypeptide encoded by a non-host
`DNA molecule is a "heterologous" peptide or polypeptide.
`
`[0038] An "isolated polypeptide" is a polypeptide that is
`essentially free from contaminating cellular components,
`such as carbohydrate, lipid, or other proteinaceous impuri(cid:173)
`ties associated with the polypeptide in nature. Typically, a
`preparation of isolated polypeptide contains the polypeptide
`in a highly purified form, i.e., at least about 80% pure, at
`least about 90% pure, at least about 95% pure, greater than
`95% pure, or greater than 99% pure. One way to show that
`a particular protein preparation contains an isolated polypep(cid:173)
`tide is by the appearance of a single band following sodium
`dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of
`the protein preparation and Coomassie Brilliant Blue stain(cid:173)
`ing of the gel. However, the term "isolated" does not exclude
`the presence of the same polypeptide in alternative physical
`forms, such as dimers or alternatively glycosylated or
`derivatized forms.
`
`[0039] The terms "amino-terminal" or "N-terminal" and
`"carboxyl-terminal" or "C-terminal" are used herein to
`denote positions within polypeptides. Where the context
`allows, these terms are used with reference to a particular
`sequence or portion of a polypeptide to denote proximity or
`relative position. For example, a certain sequence positioned
`carboxyl-terminal to a reference sequence within a polypep(cid:173)
`tide is located proximal to the carboxyl terminus of the
`reference sequence, but is not necessarily at the carboxyl
`terminus of the complete polypeptide.
`
`[0040] A "fusion protein" is a hybrid protein expressed by
`a nucleic acid molecule comprising nucleotide sequences of
`at least two genes.
`
`[0041] The term "affinity tag" is used herein to denote a
`polypeptide segment that can be attached to a second
`polypeptide to provide for purification or detection of the
`second polypeptide or provide sites for attachment of the
`second polypeptide to a substrate. In principal, any peptide
`or protein for which an antibody or other specific binding
`agent is available can be used as an affinity tag. Affinity tags
`include a poly-histidine tract, protein A (Nilsson et al.,
`EMBO J. 4:1075 (1985); Nilsson et al., Methods Enzymol.
`198:3 (1991 )), glutathione S transferase (Smith and Johnson,
`Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et
`al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P,
`FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),
`streptavidin binding peptide, or other antigenic epitope or
`binding domain. See, in general, Ford et al., Protein Expres(cid:173)
`sion and Purification 2:95 (1991 ). DNA molecules encoding
`affinity tags are available from commercial suppliers ( e.g.,
`Pharmacia Biotech, Piscataway, N.J.).
`
`[0042] The term "isotonic" is used herein for its conven(cid:173)
`tional meaning, that is a tonicity equal to that of blood,
`
`equivalent to a 0.9% solution of NaCl. "An isotonic amount"
`of a salt is that amount required to make a solution isotonic
`or to produce an isotonic solution upon reconstitution of a
`lyophilized preparation.
`
`[0043] Concentrations are specified herein in units of
`molarity or % w/v of liquid compositions. When the com(cid:173)
`position is in the form of a lyophilized powder, the concen(cid:173)
`trations of the respective components will be such as to
`provide the specified concentration on reconstitution of the
`powder.
`
`[0044] Due to the imprecision of standard analytical meth(cid:173)
`ods, molecular weights and lengths of polymers are under(cid:173)
`stood to be approximate values. When such a value is
`expressed as "about" X or "approximately" X, the stated
`value of X will be understood to be accurate to ±10%.
`
`Expression of Recombinant FGF18 or TRFGF18
`
`[0045] The present invention provides expression vectors
`and methods for producing recombinant FGF18 protein
`from a prokaryotic host. FGF18 was previously designated
`zfgf5, and is fully described in commonly assigned U.S. Pat.
`Nos. 5,989,866 6,518,236 and 6,352,971, incorporated
`herein by reference. In particular, the expression vectors and
`methods of the present invention comprise an E. coli expres(cid:173)
`sion system for the large scale production of FGF18 or
`trFGF18 utilizing the FGF18 or trFGF18 coding sequence
`with specific changes in nucleotides in order to optimize
`codons and mRNA secondary structure for translation in E.
`coli. Using the expression vectors and methods of the
`present invention, the FGF18 or trFGF18 gene was pro(cid:173)
`duced in E. coli to a level of greater than 1 g/L in fed batch
`fermentation. The present inventors found that use of the E.
`coli OmpT protease deficient strains like, for example,
`UT5600, as a production host overcame stability problems
`with FGF 18 or trFGF 18. In addition to full length FGF 18, a
`different polypeptide can be produced, truncated FGF18
`(trFGF18) that is the FGF18 coding sequence with a codon
`encoding an N-terminal Met added at the 5' end of the
`polynucleotide sequence at amino acid 27 (Glu) and trun(cid:173)
`cated at amino acid 196 (Lys). The trFGF18 sequence is
`SEQ ID NO: 5. Using the expression vectors described
`herein significantly improved the yield of recombinant pro(cid:173)
`tein recovered from the bacteria. In another embodiment, to
`facilitate the development of high cell density fed-batch
`fermentation, another E. coli strain, W3110, was selected as
`a host for the large-scale production of FGF18 or trFGF18.
`This host strain is non-pathogenic and can grow to high cell
`density in minimally defined fermentation media. The pro(cid:173)
`ductivity ofFGF18 or trFGF18 in E.coli strain W3110 was
`comparable to that obtained in E. coli strain UT5600 when
`produced in shaker flask and batch fermentations.
`
`[0046] The present invention also provides methods for
`recovering recombinant FGF18 or trFGF18 protein from a
`prokaryotic host when the protein is expressed by the host
`and found within the host cell as an unglycosylated,
`insoluble inclusion body. When the prokaryotic cell is lysed
`to isolate the inclusion bodies (also called refractile bodies),
`the inclusion bodies are aggregates of FGF18 or trFGF18.
`Therefore, the inclusion bodies must be disassociated and
`dissolved to isolate the FGF18 or trFGF18 protein, and
`generally this requires the use of a denaturing chaotropic
`solvent, resulting in recovering a polypeptide that must be
`refolded to have significant biological activity. Once the
`
`7 of 48
`
`Fresenius Kabi
`Exhibit 1004
`
`

`

`US 2006/0172384 Al
`
`Aug. 3, 2006
`
`5
`
`FGF18 or trFGF18 protein is refolded, the protein must be
`captured and purified. Thus, the present invention provides
`for methods for isolating insoluble FGF18 or trFGF18
`protein from prokaryotic cells, dissolving the insoluble
`protein material in a chaotropic solvent, diluting the chao(cid:173)
`tropic solvent in such a manner that the FGF18 or trFGF18
`protein is refolded and isolated. The present invention also
`includes methods for capturing the renatured FGF18 or
`trFGF18 from the dilute refold buffer using cation exchange
`chromatography, and purifying the refolded FGF18 or
`trFGF18 protein using hydrophobic interaction chromatog(cid:173)
`raphy. Further purification can be achieved using anion
`exchange in binding assays using an FGF 18 receptor and the
`like.
`[0047] The human FGF18 gene encodes a polypeptide of
`207 amino acids. The full length sequence includes a signal
`peptide of 27 amino acids, as shown in SEQ ID NOS: 3 and
`4, and a mature protein of 180 amino acids comprising
`residue 28 (Glu) to residue 207 (Ala). Truncated FGF18
`(trFGF18) comprises the fragment from residue 28 (Glu) to
`196 (Lys) with an added N-terminal Met. The nucleotide
`sequence of SEQ ID NO: 3 (SEQ ID NO: 1 nucleotides
`1495-2040) and SEQ ID NO:5 (SEQ ID NO: 2 nucleotides
`1494-2007) show codon optimized sequences that fall
`within the scope of the present invention.
`[0048] Production of recombinant human FGF18 or
`trFGF18 which utilized a mannnalian expression system
`produced approximately 20 mg/L of protein. Therefore, a
`more cost effective expression system was desirable for
`large-scale production of FGF18 or trFGF18. The E. coli
`system was found to be a better alternative for large-scale
`production. Expression in E. coli offers numerous advan(cid:173)
`tages over other expression systems, particularly low devel(cid:173)
`opment costs and high production yields.
`[0049] Recombinant FGF18 or trFGF18 expressed in E.
`coli was isolated as insoluble inclusion bodies after cell
`breakage. In most cases inclusion bodies needed to be
`solublized in denaturing chaotropic solvent and the protein
`refolded by dilution of the chaotropic agent followed by
`purification. Proteins vary a great deal with respect to their
`optimal refolding environment. Factors that can affect the
`recovery of properly folded and biologically active material
`include: initial protein concentration, oxidative state, pH,
`excipients, salts, detergents, termperature, mode of refolding
`buffer addition and the like.
`[0050] Examination of the codons used in the human
`FGF18 c