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`O)
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`Europadisches Patentamt
`European Patent Office
`Office européen des brevets
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`I
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`(i) Publication number: 0 657 466 A1
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`42)
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`EUROPEAN PATENT APPLICATION
`
`@1) Application number : 94307839.4
`
`@2) Date offiling : 25.10.94
`
`61) Int. cL®: CO7K 1/113, C12N 9/64,
`BO1D 61/14, CO7B 63/00
`
`Priority : 10.11.93 US 150632
`
`Date of publication of application :
`14.06.95 Bulletin 95/24
`
`Designated Contracting States:
`AT BE CH DE DK ESFR GB GRIE IT LI LU NL
`PT SE
`
`Applicant : PFIZER INC.
`235 East 42nd Street
`New York, N.Y. 10017 (US)
`
`
`
`(72) Inventor : Crawford, JamesG.
`4351 East Dallas Drive
`Terre Haute, Indiana 47802 (US)
`Inventor : Stober, Stanley R.
`23 River Drive
`Gales Ferry, Connecticut 06335 (US)
`
`Representative : Moore, James William, Dr.
`Pfizer Limited
`Ramsgate Road
`Sandwich Kent CT13 9NJ (GB)
`
`A processfor refolding of (pro-)chymosin, comprising recycling of urea.
`
`An ultrafiltration process permits recycle of a
`substantial portion of chaotrope used in
`iso-
`lation of protein produced by a host organism
`transformed with a vector.
`
`EP0657466A1
`
`Jouve, 18, rue Saint-Denis, 75001 PARIS
`
`Amgen Exhibit 2017
`Apotex Inc. et al. v. Amgen Inc. et al., IPR2016-01542
`Page 1
`
`

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`1
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`EP 0 657 466 A1
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`TECHNICAL FIELD OF THE INVENTION
`
`This invention relates to a process for the recy-
`cling of denaturing or solubilizing agents known as
`chaotropes which are used to solubilize inclusion
`bodies or insoluble proteins obtained from bacteria
`using recombinant DNAbiotechnology.
`
`BACKGROUND OF THE INVENTION
`
`Purification schemesfor proteins produced by re-
`combinant technology usually involve isolation of in-
`soluble heterologous proteins, also known asinclu-
`sion bodies, followed by denaturation and solvation
`and subsequent renaturation to obtain the biologically
`active protein. Denaturation is often accomplished by
`treatment with concentrated solutions of solubilizing
`agents known as chaotropes; renaturation into the
`biologically active form of the protein occurs sponta-
`neously when the chaotrope is removed ordiluted.
`U.S. Patent No. 5,082,775 describes a process
`for chymosin production which includes the steps of
`solubilization of inclusion bodies with 8M urea, dilu-
`tion with an alkaline pH buffer and subsequent neu-
`tralization which results in rapid renaturation.
`International Patent No. WO 83/04418 describes
`
`a processfor producing chymosin in whichthe insolu-
`ble form of the chymosin precursoris reversibly dena-
`tured with 7M urea or 6M guanidine hydrochloride.
`The urea or guanidine hydrochloride is then removed
`by dialysis after which the protein returnsto a confor-
`mation capable of being converted to active chymo-
`sin.
`
`Both of the above procedures when adapted toa
`commercial processes produce a dilute urea waste
`solution which results in a difficult and expensive dis-
`posal problem.
`U.S. Patent No. 5,202,239 discusses solubiliza-
`tion of inclusion bodies containing atrial natriuretic
`peptide (ANP) with 6M urea. The purified fusion pro-
`tein was shownto contain proteolytic enzymes from
`E. coli. These were cleared by contact with immobil-
`ized staph V8. ANP wasseparated from the high mo-
`lecular weight cleavage products by ultrafiltration
`over an Amicon YM10 membrane with a 10,000 mo-
`lecular weight cut off. In addition to ANP,thefiltrate
`stream contained the N-terminal peptide and urea
`from the cleavage reaction. ANP was separated by
`cation exchange chromatography or gelfiltration
`chromatography. This reference also describes a
`numberof other peptide substances which are formed
`as fusion proteins and must undergo a solubilization
`step during processing. These include urogastrone,
`proinsulin, and epidermal growth factor.
`
`SUMMARYOF THE INVENTION
`
`This invention provides an improved process for
`
`the preparation of chymosin or prochymosin in which
`an insoluble form of said chymosin or prochymosin is
`producedby E. Coli having a gene codingfor said chy-
`mosin or prochymosin wherein said insoluble form is
`solubilized by reversibly denaturing said insoluble
`form with urea to produce a soluble form of said chy-
`mosin or prochymosin, and removing said urea and
`allowing said solubilized form to renature thereby pro-
`ducing the soluble native form of said chymosin or
`prochymosin, wherein the improvement comprises:
`a) removing at least 50% of said urea from said
`solubilized denatured form of said chymosin or
`prochymosin by passing said urea through an ul-
`trafiltration membrane; and
`b) adding said urea removedin step a) to supply
`an effective denaturing amount of urea to an ad-
`ditional quantity of said insoluble form of an ad-
`ditional quantity of said chymosin or prochymosin
`to producesaid soluble form of said chymosin or
`prochymosin; and periodically cleaning said
`membraneby:
`c) flushing the system at a high flow rate of at
`least three holdup volumes of pH 10.3 - 10.5 di-
`lution buffer;
`d)flushing at a high flow rate with at least 3 hold-
`up volumesof 50°-54°C deionized water;
`e) repeating step d) maintaining the pH at 9.5-
`11.0;
`f) adding sodium hypochlorite solution to a free
`chlorine level of 180-200 ppm;
`g) recirculating said hypochlorite solution for 20
`to 60 minutes; and
`h) flushing with deionized water until said chlor-
`ine level is less than 0.15ppm.
`The process of this invention may be used in a
`conventional batch processandis easily adaptable to
`use in a continuous process suchas described in Uni-
`ted States Patent No. 4,999,422.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Recombinant DNAprocedures havecreated spe-
`cial problems in the purification of certain proteins
`from cell extracts and particularly in recovering such
`proteins in useable forms. These recent develop-
`ments in recombinant DNA proceduresallow the syn-
`thesis of foreign proteins in host microorganisms,
`such as bacterial, yeast and animalcells. This is ac-
`complished by transforming a hostcell with a DNAse-
`quence coding for the expression of a foreign protein.
`When thelevel of expression of the DNAsequencein
`a transformed hostcell is high, a large amount of the
`foreign protein is produced within the hostcell. Typi-
`cally the cell sequesters most of these foreign pro-
`teins in inclusion bodies within the cytoplasm of the
`cell. The proteins sequestered in this mannerarein
`the form of insoluble protein aggregates primarily
`composed of many monomersof the foreign protein
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`Page 2
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`EP 0 657 466 A1
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`4
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`bound together, typically through hydrophobic inter-
`actions. In this insoluble, aggregate form, the protein
`is typically not in its preferred biologically active con-
`formation such that the protein is capable of effecting
`its intended in vivo physiological responses.
`Thus, purification of the protein in a biologically
`active form from the insoluble aggregates requires a
`means ofsolubilizing the protein aggregates in such
`a wayto preserve,or to enable ultimate recoveryof,
`the protein in a biologically active conformation.
`The initial step in recovering the inclusion body
`contained proteins from transformed cells generally
`involves breakage of the cells through a combination
`of enzyme treatment and mechanical disruption to re-
`lease the inclusion bodies. Because the protein ag-
`gregates containedin the inclusion bodies are insolu-
`ble, centrifugation of the resulting cellular material
`producesa pellet containing a significant amount of
`the foreign protein, still in the form of insoluble aggre-
`gates. The pellet also contains lipids,
`lipopolysac-
`charides and traces of nucleic acids.
`
`The next step typically used to recoverthe protein
`is to "solubilize" the insoluble protein aggregates.
`This may be accomplished by treating the membrane
`pellet with a strong chaotrope, e.g., guanidine hydro-
`chloride, to denature and dissolve the protein. “Solu-
`bilization" may also be effected using less stringent
`chaotropes, e.g., urea that "solubilize" but do not
`completely denature the desired protein. See e.g.,
`U.S. Pat. No. 4,652,630. The workerof ordinary skill
`will understand how to select a suitable chaotrope for
`his particular process.
`Solubilizing agents or chaotropesdisrupt the pro-
`tein’s conformation factors and “unfold" the protein to
`a degree that depends onthe strength of the solubil-
`izing agent. The greater the extent of the unfolding,
`the less degree of biological activity the protein likely
`displays. The resulting solubilized protein solution ob-
`tained after one or more of the above-described "sol-
`
`ubilizations", thus comprises the foreign protein in
`somestage of unfolding, depending on the particular
`solubilizing treatment employed. To obtain a biologi-
`cally active conformation of the desired protein,
`it
`mustthus be "refolded". The solubilized protein solu-
`tion also contains other soluble or solubilized phos-
`pholipids, lipopolysaccharides, proteins, and nucleic
`acids from the inclusion body and insoluble cellular
`debris. These must, of course, be removedeither be-
`fore or after refolding of the foreign protein.
`Atypical refolding method used to refold solubil-
`ized proteins involves diluting out the solubilizing
`agent with a large volume ofdiluent, generally a buf-
`fer. When the concentration of solubilizing agentis re-
`duced to a dilution level where the protein’s confor-
`mation factors begin to reassert themselves, the pro-
`tein spontaneously refolds into a soluble, biologically
`active conformation. Depending on the protein, once
`this ’optimal dilution level" is reached, refolding be-
`
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`gins to occur within seconds or may take several min-
`utes or longer.
`Typically, the dilution is carried out in one step by
`mixing the solubilized protein solution with a diluent
`in a amount necessary to reach the optimal level of
`dilution. This dilution method is known as a "batch"di-
`
`lution, drawing its name from the procedure of adding
`the diluent in one operation to the solubilized protein
`solution. Utilization of this batch methodfor refolding
`a solubilized protein has several disadvantages
`which are magnified when the refolding is carried out
`with the large volumes used in commercial scale pur-
`ification procedures.
`Because a solubilized protein solution generally
`has to be diluted with manytimesits volumeofdiluent
`to achieve at least some degreeofrefolding, the total
`volumesbeing handled at once in commercial protein
`purification methods can be very large. This process
`results in large volumesof dilute chaotrope solution
`which present a serious waste disposal problem.
`For example, approximately 5-8M urea is re-
`quired to solubilize the inclusion bodies (IB) contain-
`ing the desired protein produced by bacteria. This sol-
`ution must be diluted by 30-50 fold to allow renatura-
`tion to occur. The resulting large volumeofdilute urea
`presentsa significant challenge to a waste treatment
`facility as the volume of the product increases.
`There has been a long felt need for a process to
`recycle a concentrated solution of chaotrope such as
`urea whichis used to solubilize inclusion bodiesorin-
`
`soluble proteins obtained from bacteria or othercells
`using recombinant DNA technology.
`We have found that a concentrated chaotrope
`solution may be separated from the above-described
`protein byultrafiltration and subsequently recycled in
`a batch or continuous process. For example, urea,
`with a molecular weight of 60 is very small compared
`to the desired protein; the ultrafiltration membrane
`must be chosen with a molecular weight cut off
`(MWCO)whichis larger than urea and smaller than
`the protein. The membrane mustbestable at high pH,
`about 10-11 for the prochymosin process described
`herein below. The workerof ordinary skill will be able
`to select an ultrafiltration membrane which is suited
`
`to his particular process.
`
`
`SUMMARY OF PROCHYMOSIN PROCESSWITH
`UREA RECYCLE
`
`Prochymosin inclusion bodies (IB) are dissolved
`in a 5 to 8M urea solution and then adjusted to an al-
`kaline pH to promote denaturation of the protein. The
`denatured solution is then concentrated via recircu-
`
`lation of the material to an ultrafiltration (UF) system.
`The small molecules (urea and water) passto the per-
`meate. The permeate then is utilized to supply urea
`to dissolve IB’s in a subsequent prochymosin disso-
`lution batch. The UF membranesare cleaned after
`
`Page 3
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`EP 0 657 466 A1
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`6
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`every batch via a hot waterflush, a recirculation hot
`water alkaline pH wash, a recirculating hot bleach
`high pH wash, and several more water flushes.
`
`MEMBRANE SELECTION
`
`In studies with prochymosin, MW about 40,000,
`the following membraneswereinvestigated for the re-
`cycle of the chaotrope urea.
`
`Koch/Abcor
`
`HFK-131-VSV; 10,000 MWCO, polysulfone
`4 inch spiral
`2 inch spiral
`HFK-131-VSV XL-1000, 10,000 MWCO, poly-
`sulfone
`
`4 inch spiral
`MSD-328-VS V; 5,000 MWCO, polysulfone
`4 inch spiral
`Millipore PTGC; 10,000 MWCO, polysulfone
`2 inch spiral
`Millipore PLGC; 10,000 MWCO. celluloric
`2 inch spiral
`Millipore PTTK; 30,000 MWCO, polysulfone
`2 inch spiral
`IWT Membralox P19-40-200A-Z-E; 20,000
`MWCO, ceramic zirconium oxide membrane
`The preferred membrane for prochymosin is
`Koch/Abcor HFK-131-VSV, 10,000 MWCO, polysul-
`fone, 4 inch spiral (Koch Membrane Systems, Inc.,
`850 Main Street, Wilmington, MA 01887). A worker of
`ordinaryskill enabled by this disclosure would be able
`to select a membraneforhis or her particular system
`based on considerations such as chaotropeflux, re-
`tention of the protein and a repeatable and practical
`membranecleaning procedure.
`In the prochymosin process, the ultrafiltration
`(U/F) feed contains about 1% suspendedsolids. This
`is a low enough solid content to permit use of spiral
`U/F systems whichoffer the advantage of packing the
`greatest membrane surfaceareainto the least holdup
`volume. The spiral design and resultant high mem-
`brane area to holdup volumeratio facilitates a higher
`batch concentration factor which is an important de-
`sign issue.
`
`MEMBRANE CLEANING
`
`Aninclusion body (IB) dissolution stream poten-
`tially causes severe and rapid (20-50 minutes) mem-
`brane fouling which is cumulative from run to run with-
`out proper cleaning. With the prochymosin batch
`process, the maximum run time was 60 minutes be-
`fore cleaning was required.
`At the end of the batch concentration run, the
`concentrate is drained and pumped from the U/F sys-
`tem to the renaturation dilution tank. About 10-15 %
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`of the concentrate typically remains behind in the U/F
`system and recycle tank adhering to the membrane
`and piping surfaces. To effectively remove this mate-
`rial from the U/F system and not lose the associated
`product, the system is flushed at high flow rate (350
`gpm in a 1900 ft? system) with at least three holdup
`volumes(a total of about 180 gallons) of pH 10.4 di-
`lution buffer. This flushis directed from the CIP (clean
`in place) tank through the U/F system to the associ-
`ated dissolution tank and then to the renaturation
`tank.
`
`It is important to remove as muchof the product
`at nearly instantaneous dilution rates to achieve the
`best renaturation yield possible. Aslow dilution caus-
`es extreme and rapid loss of enzymeactivity. Also,
`adequate flushing is important prior to starting the
`cleaning cycle since the consumption of chemical
`cleaning agents is very much a function of how well
`the system is flushed. The next flush is made with hot
`50-54°C deionized water (DIW) to the waste treat-
`mentfacility. At least three holdup volumes are rec-
`ommended again at the high flow rate. The conduc-
`tivity of the effluent from this flush should approach
`the conductivity of the feed DIW. Next the CIP tankis
`filled with 50-54°C DIW.Recirculation of this hot DIW
`
`is initiated and the pH is adjusted to 10.5 with sodium
`hydroxide. The system is recirculated (retentate and
`permeate recycled to the CIP tank) for about 20 min-
`utes while maintaining temperature and pH and then
`the system is drained to waste treatment.
`The systemis then refilled with 50-54°C DIW and
`again the pH is adjusted to 10.5 with sodium hydrox-
`ide while the system is recirculating through the U/F.
`Oncethe pH and temperature have stabilized, bleach
`is added until a free chlorine level of 180-200 ppm is
`reached. Typical bleach consumptionis estimated at
`20 gallons of 5% bleach for each cleaning of a 1900
`Ft2 commercial system. Higher bleach concentrations
`and better flushing will reduce the volume of bleach
`consumed. The bleach is added in increments and
`
`monitored by measuring the free chlorine level. The
`free chlorine will be consumedasit oxidizes the gel
`layer on the membranesurface and thefree chlorine
`level will drop. The pH will also drop during thefirst
`few bleach additions. When the pH doesnot drop dur-
`ing the bleach addition, it is a good indicator that the
`membraneis nearly clean. The liquid in the CIP tank
`will becomeclearer (turbidity will decrease) when the
`membranesare clean. The flux rate on DIW will in-
`
`crease dramatically during the bleach treatment and
`approach flux rates of 80-100 GFD at TMP of 42.5
`psig at 50-55C. The bleachis recirculated for a mini-
`mum of 20 minutes and a maximum of 60 minutes.
`
`Membranelife is adversely effected by free chlorine
`levels above 200ppm andpH greater than 11 and tem-
`perature greater than 57C. The length of time at any
`of these conditions should be tightly monitored and
`controlled. Once the membranes are clean,
`the
`
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`EP 0 657 466 A1
`
`8
`
`bleach solution is drained to waste treatment. The
`
`C. ULTRAFILTRATION
`
`system is then flushed with at least three holdup vol-
`umes of 50-54C DIW until a free chlorine level of less
`
`than .15 ppm is measured. The CIP tank is thenfilled
`with ambient temperature DIW and the system recir-
`culated. This cools the system downpriorto filling
`with the next batch of feed stock and allows for the
`
`checking of the DIW flux which is used to benchmark
`the effectivenessof the cleaning cycle. The system is
`drained of all DIW just before introducing the next
`feed stock.It is very important that as much water be
`drained from the system as possible justpriorto filling
`the system with feed, since the membrane must be
`kept wet and the water balance on the urea recycle
`process constrains the amountof permeate that can
`be recycled.
`
`EXAMPLE1
`
`
`A. PRODUCTION OF PROCHYMOSIN
`INCLUSION BODIES
`
`The preparation of prochymosin from genetically
`modified bacteria is well knownin the art. See, for ex-
`ample International Patent WO 83/04418, and United
`States Patent No. 4,977,248.
`
`Briefly, E.coli is grownin a liquid nutrient medium,
`then pelleted by centrifugation. The pelleted cells
`are resuspended in a small volume of buffer and
`burst open, releasing the prochymosin in the form
`of densely packed insoluble aggregates (IB’s). The
`aggregates are recovered by a second
`centrifugation step, while most of the rest of the
`cellular protein and debris remain in suspension.
`The prochymosin is thus rapidly purified from the
`bulk of cellular material. The aggregates may be
`treated with acid to kill residual cells and to
`
`degrade residual DNA. The prochymosin
`aggregatesare then solubilized by denaturation in
`an alkaline urea solution.
`
`B. PROCHYMOSIN FEED TO ULTRAFILTRATION
`UNIT
`
`To 1793 grams of undiluted inclusion body sus-
`pension (IB) were added 4440 g of a 10.4M urea sol-
`ution containing 5.8 g of disodium phosphate. This
`solution is saturated with respect to urea and has a
`density of 1.155-1.159 at 23°C. The above suspen-
`sion wasstirred for 90 minutes or until all IB’s dis-
`
`solved. Caustic was then added to increase the pH to
`10.3-10.7. This procedure produces a volume of
`about 5.5 | with a density of 1.129. This liquid is fed
`to the U/F system and concentrated by a factor of 2.5-
`3.5.
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`Flux and yield data were obtained on a
`Koch/Abcorpilot plant U/F unit that was connected to
`the dischargeline of the IB dissolution tank. Thepilot
`plant consisted of a centrifugal pump capable of de-
`livering a flow of 35 gpm at 64 psig on plant water, a
`250 L. recycle tank, and two standard Koch/Abcor
`four inch diameter spiral housings connected in ser-
`ies. A 200 L permeate surge tank was positioned
`above the pilot unit to allow for collection of the per-
`meate and easy recombination of the permeate via
`gravity flow with the concentrate. Retention time did
`not exceed 75 minutes. Twelve pilot concentration
`runs and eleven cleaning runs were madeto evaluate
`the flux and retention capabilities of the HFK-131-
`VSV 10,000 MWCO Koch/Abcor polysulfone mem-
`brane. Slip stream runs demonstrated a repeatable
`membrane cleaning process and that a centrifugal
`pump can beusedin this application with concentra-
`tion factors as high as 3.3X.
`
`D. DISSOLUTION RECYCLE
`
`To 3343 gmsof recycle permeate that has a den-
`sity of about 1.116 and a urea concentration of about
`7.3M was added 5.8 gmsof solid disodium phosphate
`followed by 1100 gmsof solid urea. The mixture was
`heated at 23°C to dissolve the solid urea in the per-
`meate. This generated a saturated solution of about
`10.4 M urea with a density of 1.157-1.159 at 23°C.
`Running with a saturated liquid urea feed to the 18
`dissolution is a good wayto control the urea concen-
`tration from batch to batch. To 1793 gms of undiluted
`IB’s was added the 10.4 M liquid urea batch prepared
`above. The mixture wasstirred at 20°C for 90 minutes
`until all IB’s were dissolved. Caustic was then added
`
`to increase the pH to 10.3-10.7. (A slightly higher pH
`range 10.3-10.7 is recommended to improve yield.)
`This procedure generatesa dissolution with a volume
`of about 5.5 L that has a density of 1.129. This mate-
`rial is then fed to the U/F system and concentrated by
`a factor of 2.5-3.5. The overall average yield of pro-
`chymosin for ten iterations was 93.9%.
`
`Claims
`
`1. An improved process for the preparation of chy-
`mosin or prochymosin in which an insoluble form
`of said chymosin or prochymosin is produced by
`E. Coli having a gene coding for said chymosin or
`prochymosin wherein said insoluble form is solu-
`bilized by reversibly denaturing said insoluble
`form with urea to produce a soluble form of said
`chymosin or prochymosin, and removing said
`urea and allowing said solubilized form to rena-
`ture thereby producing the soluble native form of
`
`Page 5
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`9
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`EP 0 657 466 A1
`
`10
`
`said chymosin or prochymosin, wherein the im-
`provement comprises:
`a) removing at least 50% of said urea from
`said solubilized denatured form of said chy-
`mosin or prochymosin by passing said urea
`through an ultrafiltration membrane; and
`b) adding said urea removed in step a) to sup-
`ply an effective denaturing amountof urea to
`an additional quantity of said insoluble form of
`an additional quantity of said chymosin or pro-
`chymosin to produce said soluble form of said
`chymosin or prochymosin; and periodically
`cleaning said membrane by:
`c) flushing the system at a high flow rate of at
`least three holdup volumes of pH 10.3 - 10.5
`dilution buffer;
`d) flushing at a high flow rate with at least 3
`holdup volumes of 50°-54°C deionized water;
`e) repeating step d) maintaining the pH at 9.5-
`11.0;
`f) adding sodium hypochlorite solution to a
`free chlorine level of 180-200 ppm;
`g) recirculating said hypochlorite solution for
`20 to 60 minutes; and
`h) flushing with deionized water until said
`chlorine level is less than 0.15ppm.
`
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`EP 0 657 466 A1
`
`European Patent
`Office
`
`EUROPEAN SEARCH REPORT
`
`Application Number
`EP 94 30 7839
`
`
`
`DOCUMENTS CONSIDERED TO BE RELEVANT
`
`Catezo
`Citation of documentwith indication, where appropriate,
`
`gory
`of relevant passages
`
`Relevant
`to claim
`
`CLASSIFICATION OF THE
`APPLICATION(Int.Cl.6)
`
`A
`
`A
`
`DD-A-146 751 (VEB STICKSTOFFWERKE
`PIESTERITZ) 4 March 1981
`---
`‘Protein Folding’
`HERMANN, R.
`1993 , THE EUROPEAN PATENT OFFICE , THE
`HAGUE,
`ISBN 90-9006173-8, PAGES 1-6 AND
`77-79
`
`C07K1/113
`€12N9/64
`BO1D61/14
`C07B63/00
`
`
`
`TECHNICAL FIELDS
`SEARCHED—(int.C1.6)
`
`CO7K
`CO7B
`C12N
`BO1D
`
`
`
`
`
`The present search report has been drawn up for all claims
`Examiner
`Place of search
`Date of compiction of the search
`Hermann, R
`MUNICH
`24 January
`1995
`CATEGORYOF CITED DOCUMENTS
`T : theory or principle underlying the invention
`E ; earlier patent document, but published on, or
`X : particularly relevant if taken alone
`after the filing date
`Y: particularly relevant if combined with another
`D : documentcited in the application
`document of the same category
`L : documentcited for other reasons
`A‘technological background=—aasescesensvnruvovesnessesusensosoneraseneanenseonesesosenssnsnesesesesonecauosvosarensvoresessaveoes
`c ; eeedite scosure
`& : member of the same patent family, corresponding
`document
`
`
`
`
`
`
`EPOFORM150303.82(POSCO1)
`
`Page 7
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