(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2005/0209441 A1
`(43) Pub. Date:
`Sep. 22, 2005
`Lile
`
`US 20050209441A1
`
`(54) PROCESS FOR PROMOTING PROPER
`FOLDING OF HUMAN SERUM ALBUMIN
`USING A HUMAN SERUM ALBUMIN
`LIGAND
`
`(76) Inventor: Jackson D. Lile, Boulder, CO (US)
`
`Correspondence Address:
`HOLLAND & HART, LLP
`555 17TH STREET, SUITE 3200
`DENVER, CO 80201 (US)
`
`(21) Appl. No.:
`
`11/085,291
`
`(22) Filed:
`
`Mar. 21, 2005
`
`Related US. Application Data
`
`(60) Provisional application No. 60/555,450, ?led on Mar.
`22, 2004.
`
`Publication Classi?cation
`
`(51) Int. Cl? ................................................ .. C07K 14/765
`(52) Us. 01. ............................................................ ..530/363
`
`(57)
`
`ABSTRACT
`
`The present invention is a process for refolding and rena
`turing human serum albumin protein to substantially native
`conformation by the addition of a human serum albumin
`refolding ligand to a solution containing the protein under
`conditions conducive to refolding of the protein.
`
`Amgen Exhibit 2037
`Apotex Inc. et al. v. Amgen Inc. et al., IPR2016-01542
`Page 1
`
`

`

`US 2005/0209441 A1
`
`Sep. 22, 2005
`
`PROCESS FOR PROMOTING PROPER FOLDING
`OF HUMAN SERUM ALBUMIN USING A HUMAN
`SERUM ALBUMIN LIGAND
`
`CROSS REFERENCE TO RELATED
`APPLICATION
`
`[0001] This application claims the bene?t of the ?ling date
`of US. Provisional Patent Application Ser. No. 60/555,450,
`?led Mar. 22, 2004.
`
`TECHNICAL FIELD OF THE INVENTION
`
`[0002] The present invention relates to methods for manu
`facturing recombinant serum albumin proteins, and particu
`larly to the ?eld of refolding recombinant human serum
`albumin proteins.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Human serum albumin (hereinafter referred to sim
`ply as HSA) is the most abundant protein contained in
`plasma. It is produced in the liver, and contributes to the
`maintenance of osmotic pressure in blood and binds to
`nutrients and metabolites to thereby transport these sub
`stances. HSA has been utiliZed therapeutically in the treat
`ment of such indications as hypoalbuminemia (caused by an
`albumin loss or reduction in albumin synthesis) and hem
`orrhagic shock.
`[0004] Currently, HSA is produced primarily as a frac
`tionated product of collected blood, Which is uneconomical
`and is subject to a sporadic supply of blood. In addition,
`collected blood sometimes contains undesirable pathogenic
`substances, such as hepatitis or HIV virus. Recent advances
`in recombinant DNA techniques, hoWever, have made pos
`sible the production of various types of useful polypeptides
`by microbial host cells. Establishing techniques for the large
`scale production of HSA by recombinant methods and
`subsequent high grade puri?cation Would therefore be desir
`able.
`
`[0005] Recombinant mammalian proteins can be
`expressed in either prokaryotic host systems, such as E. coli,
`or eukaryotic host systems, such as yeast or mammalian
`cells. Eukaryotic systems possess the cellular machinery to
`ensure proper folding and most post-translational modi?ca
`tions, but typically result in loW yields and require compleX
`and eXpensive puri?cation procedures. In contrast, expres
`sion in prokaryotic systems results in much higher yields,
`but the cellular machinery necessary for proper folding of
`eukaryotic proteins is lacking.
`[0006] Some recombinant mammalian proteins Will fold
`spontaneously to the native conformation, and can therefore
`be expressed in prokaryotic systems With little dif?culty.
`Other proteins, such as large proteins and proteins With
`multiple intra-molecular disul?de bonds (covalent bonds
`betWeen tWo cysteine amino acid residues at different loca
`tions Within the protein), hoWever, are more problematic.
`Such proteins, When formed in a prokaryote cell, form large
`insoluble inclusion bodies (constituting up to 80% of the net
`Weight of the cell) comprised of an aggregation of unfolded
`or incorrectly folded proteins bound together by the forma
`tion of incorrect intra- or inter-molecular disul?de bonds.
`Such proteins, of course, are not biologically active.
`Although inclusion bodies can be readily puri?ed by cen
`
`trifugation on the basis of their relatively high density, the
`proteins forming the inclusion body are not folded in the
`native conformation and must therefore be refolded into its
`biologically active native conformation.
`
`[0007] Avariety of methods have been used to re-solubi
`liZe and refold proteins to a biologically active native
`conformation. Typically, the refolding process begins With
`Washed inclusion bodies, Which are solubiliZed With high
`concentrations of denaturants such as mercaptoethanol,
`guanidine hydrochloride or urea. The amount of aggregation
`may continue to increase With time if the protein is alloWed
`to remain in the denaturant, and incorrect disul?de bonds
`may sloW the refolding process or possibly generate kineti
`cally trapped intermediates that are dif?cult to reverse.
`Removal of the denaturant from the solubiliZed inclusion
`bodies by dialysis or desalting columns Will cause the
`protein to precipitate under conditions Where the native
`protein needs to be refolded. A misfolded protein solution
`can also have a very loW speci?c activity in biological
`assays.
`
`[0008] HSA is a particularly dif?cult protein to refold,
`primarily because it has 17 disul?de bonds (35 cysteine
`residues in total) that can incorrectly form in various com
`binations. Lee et al. (J. Bio. Chem. 267:14753-14758, 1992)
`disclose a process for refolding denatured/disul?de-reduced
`HSA by ?rst completely reducing the protein With glu
`tathione, and then completely oXidiZing the protein With
`oXidiZed glutathione, over a long period of about 48 hours.
`
`[0009] US. Pat. No. 6,617,133 (Noda et al.) also disclose
`a process for purifying recombinant human serum albumin
`by heating a culture medium containing rHSA and the
`rHSA-producing host cells, feeding the heated solution
`upWardly into a ?uidiZed bed in Which adsorbent particles
`are suspended to effect contacting With the adsorbent par
`ticles, and then recovering the adsorbed fraction containing
`the rHSA.
`
`[0010] Both of the above processes require lengthy peri
`ods of time to achieve proper refolding, Which is unaccept
`able for commercial production of rHSA. Accordingly, a
`rapid method of producing recombinant human serum albu
`min is desired.
`
`SUMMARY OF THE INVENTION
`
`[0011] The present invention is a process for refolding
`HSA to substantially native conformation using a human
`serum albumin ligand. More particularly, the present inven
`tion is a process for refolding and renaturing human serum
`albumin protein to substantially native conformation by the
`addition of a human serum albumin refolding ligand to a
`solution containing the protein under conditions conducive
`to refolding of the protein.
`
`[0012] In a particular embodiment of the present inven
`tion, a human serum albumin refolding ligand is added in the
`course of a process involving the folloWing three stages: (a)
`solubiliZing human serum albumin protein in a solution
`comprising a denaturant and a ?rst thiol reducing compound
`at concentrations suf?cient to disrupt formation of all dis
`ul?de bonds and form free thiols, (b) decreasing the con
`centration of the denaturant, at pH greater than about 9.5,
`adding to the solution a disul?de oXidiZing compound to a
`molar concentration suf?cient to create mild oXidiZing redoX
`
`Page 2
`
`

`

`US 2005/0209441 A1
`
`Sep. 22, 2005
`
`conditions to oxidize a portion of the free thiols and form
`mixed native and non-native disul?de bonds, and adding a
`human serum albumin refolding ligand at a time from stage
`(b) to (c); and (c) further decreasing the concentration of the
`denaturant, loWering the pH of the solution to less than 9.5,
`and adding a second thiol reducing compound to a molar
`concentration suf?cient to create mild reducing conditions to
`catalyZe interchange of the non-native disul?de bonds and
`form native disul?de bonds, thereby providing a human
`serum albumin protein having substantially native confor
`mation.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0013] Recombinant Expression of HSA
`[0014] The process of the present invention is used to
`assist in refolding human serum albumin (HSA) protein that
`is incompletely or incorrectly folded, for example, HSA that
`has been puri?ed from natural sources or, more commonly,
`HSA that has been produced by recombinant expression in
`host cells. The use of recombinant host cells to produce
`proteins is a highly cost effective method for obtaining large
`quantities of a protein for use in commercial applications.
`Recombinant HSA proteins are typically expressed in a
`suitable host, for example, eukaryotic cells (such as yeast
`and animal cells), or procaryotic cells (such as E. coli or
`other type of bacteria), using a standard expression vector
`such as a plasmid, bacteriophage or naked DNA, and the
`protein expressed from the plasmid or DNA integrated into
`the host chromosome. Expression in prokaryotic systems
`typically provides higher yields of the protein at loWer cost,
`but suffers from the disadvantage that prokaryotic systems
`lack the cellular machinery to correctly fold complex mam
`malian proteins. Some eukaryotic host systems may also not
`result in correctly folded proteins. The process of the present
`invention is therefore applicable to any method of producing
`HSA that results in an HSA protein that is not folded in the
`native conformation, but is particularly advantageous in
`promoting folding of HSA proteins expressed in prokaryotic
`systems.
`[0015] Suitable methods for recombinant expression of
`HSA are Well knoWn in the art. Bacterial strains for expres
`sion of HSA are commercially available or can be obtained
`from the American Type Culture Collection (ATCC), 10801
`University Boulevard, Manassas, Va. 20110-2209. Expres
`sion of recombinant proteins in host cells also requires
`suitable expression vectors, Which can be obtained from any
`number of sources, including the ATCC. Expression vectors
`require a promoter to insure that the DNA is expressed in the
`host, and may also include other regulatory sequences that
`affect the expression of the recombinant protein. The vector
`may also include means for detection, such as an antibiotic
`resistance marker, green ?uorescent protein tag, or antigen
`tag to facilitate in puri?cation of the recombinant protein.
`Once the DNA encoding the protein to be puri?ed is
`introduced into the host, the host is cultured under appro
`priate conditions until suf?cient amounts of recombinant
`protein are obtained.
`[0016] Preparation of HSA-producing host, production of
`rHSA by its culturing and isolation and recovery of rHSA
`from the cultured broth are all carried out in accordance With
`knoWn methods Which may be modi?ed for optimiZation.
`For example, preparation of an HSA-producing host may be
`effected using a process in Which a natural HSA gene is used
`(EP-A-73646, EP-A-79739 and EP-A-91527), a process in
`
`Which a modi?ed human serum albumin gene is used
`(EP-A-206733), a process in Which a synthetic signal
`sequence is used (EP-A-329127), a process in Which a serum
`albumin signal sequence is used (EP-A-319641), a process
`in Which a recombinant plasmid is introduced into a chro
`mosome (EP-A-399455), a process in Which hosts are fused
`(EP-A-409156), a process in Which a mutation is generated
`in a methanol containing medium, a process in Which a
`mutant AOX2 promoter is used (EP-A-506040), and a pro
`cess in Which HSA is expressed in B. subtilis (EP-A
`229712).
`[0017] Culturing of the HSA-producing host may be
`effected by each of the methods disclosed in the above
`patents, by a method in Which producer cells and the product
`are obtained in high concentrations by a fed-batch culture (a
`semi-batch culture) Which method is carried out by gradu
`ally supplying a high concentration solution of glucose,
`methanol or the like in appropriate small amounts to avoid
`high concentration substrate inhibition against the producer
`cells (JP-A-3-83595) or by a method in Which the HSA
`productivity is improved by the addition of fatty acids to the
`culture medium (EP-A-504823 and Us. Pat. No. 5,334,
`512).
`[0018] A medium usually employed in the art supple
`mented With a fatty acid having from 10 to 26 carbon atoms
`or a salt thereof can be used as a medium for culturing a
`transformed host, and culturing of the transformant can be
`carried out under knoWn conditions. The medium may be
`either synthetic or natural, but preferably is a liquid medium.
`For example, a suitable synthetic medium may be composed
`of: carbon sources, such as various saccharides; nitrogen
`sources, such as urea, ammonium salts, nitrates; trace nutri
`ents, such as various vitamins, nucleotides; and inorganic
`salts, such as of Mg, Ca, Fe, Na, K, Mn, Co and Cu. An
`illustrative example of such a medium is YNB liquid
`medium, Which consists of 0.7% Yeast Nitrogen Base
`(Difco) and 2% glucose. An illustrative example of a useful
`natural medium is YPD liquid medium, Which consists of
`1% Yeast Extract (Difco), 2% Bacto Peptone (Difco) and 2%
`glucose. The medium pH may be neutral, Weakly basic or
`Weakly acidic. In the case of a methylotrophic host, the
`medium may be further supplemented With methanol in an
`amount of approximately from 0.01 to 5%.
`
`[0019] The culturing temperature preferably ranges from
`15 to 43.degree.C. (20 to 30° C. for yeast, 20 to 37° C. for
`bacterium). The culturing period ranges from 1 to 1,000
`hours, preferably 20 to 360 hours, by means of static or
`shake culturing or batch, semi-batch or continuous culturing
`under agitation and aeration. It is desirable to prepare a seed
`culture prior to the batch culturing by means of static or
`shake culturing or batch, semi-batch or continuous culturing
`under agitation and aeration. The seed culturing may be
`carried out using the aforementioned YNB liquid medium or
`YPD liquid medium, preferably at 30° C. (for yeast) or 37°
`C. (for bacterium) and for 10 to 100 hours.
`
`[0020] US. Pat. No. 6,617,133 (Noda et al.) disclose a
`process for purifying recombinant human serum albumin by
`heating a-culture medium containing rHSA and the rHSA
`producing host cells, feeding said heated solution upWardly
`into a ?uidiZed bed in Which adsorbent particles are sus
`pended to effect contacting With the adsorbent particles and
`then recovering the adsorbed fraction containing the rHSA,
`and a composition comprising rHSA Which shoWs a A350/
`A280 ratio of beloW 0.015, When formulated into a 25%
`solution of albumin.
`
`Page 3
`
`

`

`US 2005/0209441 A1
`
`Sep. 22, 2005
`
`[0021] The production of rHSA in microorganisms has
`been disclosed in EP 330 451 and EP 361 991. Puri?cation
`techniques for rHSA have been disclosed in: WO 92/04367,
`removal of matrix-derived dye; EP 464 590, removal of
`yeast-derived colorants: and EP 319 067, alkaline precipi
`tation and subsequent application of the rHSA to a lipophilic
`phase having speci?c affinity for albumin.
`
`[0022] Once the protein has been expressed in the maxi
`mum amount, it must be separated and puri?ed from the
`bacterial host. The protein is isolated generally by lysing the
`cells, for example, by suspending in detergent, adding
`lysoZyme, and then freezing (for example, by suspending
`cells in 20 ml of TN/1% TritonTM X-100, adding 10 mg
`lysoZyme and freezing at —20° C. overnight), thaWing and
`adding DNAase to degrade all of the bacterial DNA, then
`Washing the resulting precipitate in a buffered solution. The
`precipitate is then dissolved in an appropriate solution as
`discussed beloW, for refolding.
`[0023] Refolding/Puri?cation Methods
`
`[0024] The process of the present invention provides a
`novel method for refolding HSA to substantially its native
`conformation. In one embodiment of this process, HSA is
`subjected to the folloWing processes: (A) HSA is solubiliZed
`in a solution comprising suf?cient amounts of a denaturant
`and a ?rst thiol reducing compound to disrupt formation of
`all disul?de bonds and form free thiols; (B) the concentra
`tion of the denaturant is then decreased and a suf?cient
`amount of a disul?de oxidiZing compound is added to the
`solution, at above physiological pH, suf?cient to create mild
`oxidiZing redox conditions and thereby oxidiZe a portion of
`the free thiols to form mixed native and non-native disul?de
`bonds; and (C) the concentration of the denaturant is further
`decreased, the pH of the solution is loWered, and a suf?cient
`amount of a second thiol reducing compound is added to the
`solution, suf?cient to create mild reducing conditions and
`thereby catalyZe interchange of the non-native disul?de
`bonds to native disul?de bonds. The above process provides
`a human serum albumin protein having substantially native
`conformation. In another aspect of the invention, refolding
`of HSA is further facilitated by the addition of an HSA
`refolding ligand in step B and or C.
`
`[0025] Particular embodiments of the process of the
`present invention are described beloW. Particular conditions
`relating to the embodiments described beloW can be readily
`determined by those skilled in the art of protein puri?cation
`and refolding. In particular, the temperature conditions can
`be selected as appropriate. While higher temperature con
`ditions may be used to accelerate refolding, temperature
`conditions signi?cantly greater than room temperature may
`result in degradation of the protein. In the disclosed embodi
`ments of the present invention, the temperature conditions
`may be from about 4° C. to about 45° C. In particular
`embodiments, the temperature conditions are from about 25°
`C. to about 37° C. In more particular embodiments, the
`temperature conditions are about 37° C. While particular
`preferred temperature conditions are disclosed beloW, such
`conditions may be adjusted or modi?ed, as appropriate.
`[0026] Ligand-Assisted Refolding
`[0027] The present invention relates to a process for
`refolding HSA protein utiliZing one or more HSA binding
`ligands capable of promoting refolding of HSA. As used
`
`herein, the term “HSA refolding ligand” refers to a ligand
`that binds to and promotes refolding of HSA, particular
`embodiments of Which are disclosed beloW. An HSA refold
`ing ligand is added at any time from stage B to stage C of
`the process to accelerate refolding of HSA. In particular
`embodiments, an HSA refolding ligand is added at stage B,
`so that the refolding ligand is present in both stage B and the
`subsequent stage C of the process, thereby optimiZing over
`all yield. In a particular embodiment of the present inven
`tion, HSA is refolded to substantially native conformation
`by providing human serum albumin in a solution under
`conditions conducive to refolding of human serum albumin,
`and adding a human serum albumin refolding ligand com
`prising a human serum albumin site 2 binding ligand. In
`another embodiment, HSA is refolded to substantially native
`conformation by providing human serum albumin in a
`solution comprising suf?cient amounts of a denaturant, a
`?rst thiol reducing compound, and a disul?de oxidiZing
`compound to create mild oxidiZing redox conditions and
`oxidiZe a portion of the free thiols to form mixed native and
`non-native disul?de bonds, and adding a human serum
`albumin refolding ligand comprising a human serum albu
`min site 2 binding ligand. Other embodiments of the present
`invention utiliZing an HSA refolding ligand are illustrated
`beloW in the context of a staged process for refolding HSA
`protein.
`
`[0028] Suitable HSA refolding ligands may include, for
`example, the free acids or salts of the n-alkyl C2-C14 mono
`and di-fatty acids, the n-alkyl C2-C14 mono- and di-alco
`hols; ligands that speci?cally bind HSA site 1, such as
`Warfarin, n-butyl p-aminobenZoate or Indomethacin; ligands
`that speci?cally bind HSA site 2, such as Dichlofenac,
`Ibuprofen, Naproxen, L-thyroxine, L-trytophan, and so
`forth. Compounds knoWn to bind to other sites are also
`effective as refolding ligands such as bilirubin, lithocholic
`acid, lithocholic sulfate, as Well as soluble calcium salts.
`Also, combinations of HSA refolding ligands that bind to
`different sites of the HSA protein may be used. In particular
`embodiments of the present invention, the HSA refolding
`ligand is a ligand that binds to HSA site 2.
`
`[0029] In one embodiment of the present invention, HSA
`protein is refolded using ligands capable of speci?cally
`binding HSA site 1, such as 8-anilino-1-naphthalenesulfonic
`acid, Indomethacin, n-butyl p-aminobenZoate, Warfarin,
`sodium salicylate, Tolbutamide, sodium valproate, lodipa
`mide, dansyl-L-asparagine, Sul?soxaZole, Phenol Red, Phe
`nylbutaZone, and other knoWn site 1 binding ligands. In
`order to prevent contamination of the puri?ed HSA protein
`With the above ligands, use of the above ligands to promote
`refolding is preferably accomplished by immobiliZing the
`ligands on a resin, exposing HSA to the immobiliZed ligand
`to promote refolding, and then eluting HSA from the immo
`biliZed resin in a puri?ed form.
`
`[0030] More particularly, suitable HSA refolding ligands
`include ligands that bind to HSA site 2. Particular ligands
`that are knoWn to bind to HSA site 2 include the various fatty
`alcohols, fatty acids, and salts of fatty acids. The fatty acid
`salts may be, for example, alkaline earth salts, including
`sodium salts and potassium salts, as Well as other salts, such
`as ammonium salts. In particular embodiments, the fatty
`acids salts are sodium salts.
`
`Page 4
`
`

`

`US 2005/0209441 A1
`
`Sep. 22, 2005
`
`[0031] In another embodiment of the present invention,
`HSA protein is refolded using ligands that bind to site 2 of
`HSA, such as Ibuprofen, NaproXen, Dichlofenac, L-tryp
`tophan, sodium hippurate, L-thyroXine, indole-3-acetate.
`Other site 2 binding ligands may also be used. In one
`embodiment, the site 2 ligand is Ibuprofen, NaproXen,
`Dichlofenac, L-tryptophan. In yet another embodiment, the
`site 2 ligand is L-tryptophan, a natural and essential amino
`acid that might not result in unacceptable contamination of
`the puri?ed HSA protein. Again, in order to prevent con
`tamination of the puri?ed HSA protein With the above
`ligands, use of the above ligands to promote refolding is
`preferably accomplished by immobiliZing the ligands on a
`
`(C8), heXanol (C6), pentanol (C5), butanol (C4), propanol
`(C3), and ethanol (C2). In particular embodiments, C6-C14
`alcohols are used to assist refolding. C2-C4 alcohols are
`more effective at high concentrations, Which may be of use
`for large scale manufacturing. The alcohols may be primary,
`secondary, or tertiary alcohols (With the OH group bonded
`to the primary, secondary, or tertiary carbon atom). In other
`particular embodiments, the n-alkyl alcohols are primary
`straight chain alcohols, With the OH group bonded to the
`primary carbon. Such alcohols include, for eXample, pro
`panol, butanol, and pentanol. Examples of particular alco
`hols and particular fatty acids, and preferred and optimal
`concentrations are listed beloW in Table 1.
`
`Refolding Ligand
`
`*ED50
`
`Preferred Range
`
`Optimal Concentration
`
`TABLE 1
`
`Fatty acids
`
`Na Laurate (C12)
`Na Caprate (C10)
`Na Caprylate (C8)
`Na Caproate (C6)
`Alcohols
`
`10 uM-5 mM
`45 uM
`30 uM-30 mM
`125 uM
`2 mM 500 uM-30 mM
`17 mM
`3 mM-100 mM
`
`1 mM
`10 mM
`30 mM
`>30 mM
`
`50 uM-300 uM (saturated) 300 uM (saturated)
`1-Dodecanol (C12) 230 uM
`100 uM-10 mM
`3 mM
`1-Decanol (C10)
`300 uM
`100 uM-10 mM
`3 mM
`1-Octanol (C8)
`450 uM
`1-HeXanol (C6)
`2 mM 0.5 mM-100 mM
`25 mM
`1-Pentanol (C5)
`25 mM
`5 mM-500 mM
`150 mM
`l-Butanol (C4)
`125 mM 25 mM-1 M
`500 mM
`l-Propanol (C3)
`800 mM 100 mM—1.5 M
`>1 M
`
`*ED50 for refolding is that concentration of compound Which gives 50% refolding in 30
`minutes at 37° C. With HSA at 1 mg/ml.
`
`resin, eXposing HSA to the immobiliZed ligand to promote
`refolding, and then eluting HSA from the immobiliZed resin
`in a puri?ed form.
`
`[0032] In another embodiment of the present invention,
`HSA protein is refolded using n-alkyl fatty acids or their
`alkaline earth salts capable of binding to HSA. In particular
`embodiments, the refolding ligand is an n-alkyl fatty acids
`or their alkaline earth salts capable of binding to site 2 of
`HSA. Such ligands include sodium myristate, sodium lau
`rate, sodium caprate, sodium caprylate, sodium caproate,
`sodium butyrate, and sodium acetate. In a particular embodi
`ment of the present invention, the ligand is a C8-C12 fatty
`acid, such as sodium laurate, sodium caprate and sodium
`caprylate. Examples of particular fatty acids are shoWn
`beloW in Table 1.
`[0033] In the conteXt of protein refolding, acceptable fatty
`acid salts Will generally promote refolding at concentrations
`equal to or greater than about 0.1 mM. In particular embodi
`ments of the invention, the concentration of fatty acid salts
`used to promote refolding is greater than about 1 mM, and
`more preferably greater than about 10 mM. As shoWn beloW
`in Table 1, the acceptable ranges and optimal concentrations
`Will vary according to the choice of refolding ligand. In a
`particular embodiment, sodium caprate is used as the HSA
`refolding ligand, at higher concentrations of from about 1 to
`about 10 mM.
`
`[0034] In a particular embodiment of the present inven
`tion, HSA protein is refolded using n-alkyl alcohols such as
`tetradecanol (C14), dodecanol (C12), decanol (C10), octanol
`
`[0035] In another embodiment of the present invention,
`HSA protein is refolded using ligands capable of binding
`HSA at sites other than site 1 or 2, such as bilirubin,
`lithocholic acid, lithocholic sulfate, and other compounds
`referred to bile salts.
`
`[0036] Particular compounds useful in assisting refolding
`of HSA proteins include those listed in the folloWing Table
`2. Table 2 shoWs the reported binding constants of site 1
`ligands and their observed ED50 values for refolding assis
`tance (ED50 for refolding is that concentration of compound
`Which gives 50% refolding in 30 minutes at 37° C. With HSA
`at 1 mg/ml). Table 2 shoWs that site 1 ligands With very
`similar binding constants may have very different refolding
`ED50’s.
`
`TABLE 2
`
`Site 1 Ligands
`
`ED50 for
`HSA refolding
`Very different
`refolding ED50’s
`
`Binding
`constant
`Similar binding
`constants
`
`Site 1 ligands
`
`Comparison of 4
`compounds
`
`n-butyl p-aminobenzoate
`Warfarin
`Na Salicylate
`Na Valproate
`Comparison of 2
`
`800 uM
`3 mM
`12 mM
`15 mM
`
`2.8 x 10.5
`3.4 x 10.5
`1.9 x 10.5
`2.8 x 10.5
`
`Page 5
`
`

`

`US 2005/0209441 A1
`
`Sep. 22, 2005
`
`TABLE 2-continued
`
`Site 1 Ligands
`
`Site 1 ligands
`
`compounds
`
`Indomethacin
`Sul?soxazole
`
`ED50 for
`HSA refolding
`Very different
`refolding ED50’s
`
`Binding
`constant
`Similar binding
`constants
`
`600 uM
`>30 mM
`
`1.4 x 10.6
`1 x 10.6
`
`[0037] Table 3 shows some site 2 ligands With very similar
`binding constants and very similar refolding ED50’s.
`
`TABLE 3
`
`Site 2 Ligands
`
`Comparison of
`3 compounds
`
`Ibuprofen
`Naproxen
`Dichlofenac
`
`Very similar
`refolding
`ED50’s
`
`400 uM
`300 uM
`300 uM
`
`Comparison of
`4 fatty acids
`
`Refolding ED50
`progression
`
`Na Laurate
`Na Caprate
`Na Caprylate
`Na Caproate
`
`45 uM
`125 uM
`2 mM
`17 mM
`
`Very similar
`binding
`constants
`
`2.7 x 10.6
`3.7 x 10.6
`3.3 x 10.6
`
`Binding
`constant
`progression
`
`1.1 x 10.7
`8.3 x 10.6
`1.6 x 10.6
`7 x 10.4
`
`[0038] Stage A: HSA Reduction
`[0039] In stage A of the process, HSA is denatured and
`reduced to disrupt formation of all disul?de bonds and form
`free thiols. In particular embodiments of the invention, stage
`A comprises solubiliZing HSA protein in a solution com
`prising a denaturant and a ?rst thiol reducing compound at
`concentrations suf?cient to disrupt formation of all disul?de
`bonds and form free thiols. In another embodiment, stage A
`comprises solubiliZing human serum albumin protein in a
`solution comprising a denaturant, and a ?rst thiol reducing
`compound at a molar concentration greater than the molar
`concentration of human serum albumin disul?de bonds,
`sufficient to disrupt formation of all disul?de bonds and form
`free thiols. In yet another embodiment, stage A comprises
`solubiliZing human serum albumin protein in a solution
`comprising urea, and a ?rst thiol reducing compound to a
`molar concentration greater than the molar concentration of
`human serum albumin disul?de bonds, suf?cient to disrupt
`formation of all disul?de bonds and form free thiols. The
`denaturant and ?rst thiol reducing compound may be added
`to the HSA solution in any desired order.
`
`[0040] HSA must ?rst be dissolved in an appropriate
`buffer solution that is compatible With the HSA protein and
`the particular reagents selected, Which can be readily deter
`mined by those skilled in the art of protein puri?cation and
`refolding. As used in the present invention, compatible
`buffer solutions include those using sodium bicarbonate,
`sodium borate, ammonium acetate, and so forth. In particu
`lar embodiments, sodium bicarbonate and sodium borate are
`
`used. In a more particular embodiment of the present inven
`tion, HSA is dissolved in a buffer solution of sodium
`bicarbonate, Which may contain from 5 mM to 500 mM
`sodium biocarbonate. In another embodiment, the sodium
`bicarbonate buffer solution may contain from 10 mM to 100
`mM sodium biocarbonate. In yet anther embodiment, the
`sodium bicarbonate buffer solution is about 15 mM sodium
`bicarbonate.
`
`[0041] Inclusion bodies that form during recombinant
`expression of mammalian protein, such as HSA, contain
`disul?de bonds that must be disrupted in the presence of
`reducing reagents. Accordingly, the initial buffer solution of
`the process of the present invention Will also contain a
`reducing agent to disrupt intramolecular and intermolecular
`disul?de bonds that form Within and between HSA mol
`ecules. Representative reducing agents include thiol based
`reducing agents such as dithiothreitol (DTT), dithioerythri
`tol, 2-mercaptoethanol, cysteine, cysteamine, glutathione,
`ethanethiol, l-propanethiol, 3-methyl-1-butanethiol, or a
`non-thiol based compound such as TCEP (Tris[2-carboxy
`ethyl]phosphine). In particular embodiments of the present
`invention, dithiotheritol and TCEP are used as reducing
`agents.
`
`[0042] The molar concentration of the reducing agent
`should be at least equal to the molar concentration of protein
`disul?de bonds. In particular embodiments of the present
`invention, the molar ratio of the ?rst reducing agent is
`greater than the protein disul?de concentration. In another
`embodiment, the molar ratio to the ?rst reducing agent is
`from about 1-20 times that of the protein disul?de concen
`tration. In another embodiment, the molar ratio of the
`reducing agent is from about 1-10 times that of the protein
`disul?de concentration. In yet another embodiment, the
`molar ratio of the reducing agent is about 1.5 to 3 times that
`of the protein disul?de concentration.
`
`[0043] Finally, the buffer solution used for the process of
`the present invention Will also contain a chaotropic agent to
`further assist in disrupting inter- and intra-molecular attrac
`tive forces Within and betWeen HSA molecules, and thereby
`dissolve the HSA protein into solution. Suitable chaotropic
`reagents include, for example, urea, guanidine hydrochlo
`ride, and thiocyanate. As used in the process of the present
`invention, the concentration of urea used to denature protein
`precipitates and aggregates are greater than about 3 M to
`about 10 M. In other embodiments, the concentration of urea
`if from about 5 M to about 8 M. In yet another embodiment,
`the concentration of urea is about 6 M.
`
`[0044] The pH of the buffer solution is also a factor in
`achieving successful refolding of the HSA protein. Gener
`ally, the refolding process is initiated at high pH conditions,
`suf?cient to facilitate dissolution of precipitated or aggre
`gated proteins in the presence of a reducing agent and a
`chaotrope, such as 6-8 M urea. The particular pH of a buffer
`Will generally be selected to be compatible With other steps
`in the refolding process. In the refolding process of the
`present invention, HSA is initially dissolved in a buffer
`solution at a pH suf?cient to dissolve the HSA protein in the
`presence of a reducing agent and a chaotropic agent. In the
`illustrated embodiments of the present invention, the initial
`buffer solution has a pH from about pH 3 to about pH 11,
`depending on the chosen reducing agent. In one embodi
`ment, t