0,
`
`Européiisches Patentamt
`European Patent Office
`Office européen des brevets
`
`@ Publication number: 0 657 466 A1
`
`@
`
`EUROPEAN PATENT APPLICATION
`
`@ Application number: 943073304
`
`@ Date of filing : 25.10.94
`
`@ Int. CL“: C07K 1/113, C12N 9/64,
`BO1D 61/14, C07B 63/00
`
`Priority: 10.11.93 us 150632
`
`Date of publication of application :
`14.06.95 Bulletin 95/24
`
`@ Inventor: Crawford, James G.
`4351 East Dallas Drive
`Terre Haute, Indiana 47802 (US)
`Inventor: Stober, Stanley R.
`23 River Drive
`Gales Ferry, Connecticut 06335 (US)
`
`Designated Contracting States:
`AT BE CH DE DK ES FR GB GR IE IT LI LU NL
`PT SE
`
`Representative: Moore, James William, Dr.
`Pfizer Limited
`Ramsgate Road
`Sandwich Kent CT13 9NJ (GB)
`
`@ Applicant: PFIZER INC.
`235 East 42nd Street
`New York, N.Y. 10017 (US)
`
`A process for 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 211. V. Amgen Inc. et 211., IPRZO16-01542
`Page 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 DNA biotechnology.
`
`BACKGROUND OF THE INVENTION
`
`Purification schemes for proteins produced by re-
`combinant technology usually involve isolation of in-
`soluble heterologous proteins, also known as inclu-
`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 or diluted.
`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 83l04418 describes
`
`a process for producing chymosin in which the insolu-
`ble form of the chymosin precursor is reversibly dena-
`tured with 7M urea or 6M guanidine hydrochloride.
`The urea or guanidine hydrochloride is then removed
`by dialysis after which the protein returns to a confor-
`mation capable of being converted to active chymo-
`sin.
`
`Both of the above procedures when adapted to a
`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 shown to contain proteolytic enzymes from
`E E. These were cleared by contact with immobil-
`ized staph V8. ANP was separated 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, the filtrate
`stream contained the N-terminal peptide and urea
`from the cleavage reaction. ANP was separated by
`cation exchange chromatography or gel filtration
`chromatography. This reference also describes a
`number of 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.
`
`SUMMARY OF 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
`produced by E. Coli having a gene coding for 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 removed in 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 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 di-
`lution buffer;
`d) flushing at a high flow rate with at least 3 hold-
`up 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 chlor-
`ine level is less than 0.15ppm.
`The process of this invention may be used in a
`conventional batch process and is easily adaptable to
`use in a continuous process such as described in Uni-
`ted States Patent No. 4,999,422.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Recombinant DNA procedures have created 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 procedures allow the syn-
`thesis of foreign proteins in host microorganisms,
`such as bacterial, yeast and animal cells. This is ac-
`complished by transforming a host cell with a DNA se-
`quence coding forthe expression of a foreign protein.
`When the level of expression of the DNA sequence in
`a transformed host cell is high, a large amount of the
`foreign protein is produced within the host cell. 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 manner are in
`the form of insoluble protein aggregates primarily
`composed of many monomers of the foreign protein
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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 viio physiological responses.
`Thus, purification of the protein in a biologically
`active form from the insoluble aggregates requires a
`means of solubilizing the protein aggregates in such
`a way to preserve, or to enable ultimate recovery of,
`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 treatmentand mechanical disruption to re-
`lease the inclusion bodies. Because the protein ag-
`gregates contained in the inclusion bodies are insolu-
`ble, centrifugation of the resulting cellular material
`produces a 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 nextstep 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-
`bi|ization" may also be effected using less stringent
`chaotropes, e.g., urea that "so|ubi|ize" but do not
`completely denature the desired protein. See e.g.,
`U.S. Pat. No. 4,652,630. The worker of ordinary skill
`will understand how to select a suitable chaotrope for
`his particular process.
`Solubilizing agents or chaotropes disrupt the pro-
`tein’s conformation factors and ''unfold'' the protein to
`a degree that depends on the 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 "so|-
`
`ubi|izations", thus comprises the foreign protein in
`some stage of unfolding, depending on the particular
`solubilizing treatment employed. To obtain a biologi-
`cally active conformation of the desired protein,
`it
`must thus 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 removed either 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 of diluent, generally a buf-
`fer. When the concentration ofsolubilizing agent is 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 ’optima| 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 method for 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 many times its volume ofdiluent
`to achieve at least some degree of refolding, the total
`volumes being handled at once in commercial protein
`purification methods can be very large. This process
`results in large volumes of 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 volume ofdilute urea
`presents a 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 which is used to solubilize inclusion bodies or in-
`
`soluble proteins obtained from bacteria or other cells
`using recombinant DNA technology.
`We have found that a concentrated chaotrope
`solution may be separated from the above—described
`protein by ultrafiltration 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) which is larger than urea and smaller than
`the protein. The membrane must be stable at high pH,
`about 10-11 for the prochymosin process described
`herein below. The worker of ordinary skill will be able
`to select an ultrafiltration membrane which is suited
`
`to his particular process.
`
`SUMMARY OF PROCHYMOSIN PROCESS WITH
`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) pass to the per-
`meate. The permeate then is utilized to supply urea
`to dissolve |B’s in a subsequent prochymosin disso-
`lution batch. The UF membranes are 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 water flush, 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 membranes were investigated 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). Aworker of
`ordinary skill enabled by this disclosure would be able
`to select a membrane for his or her particular system
`based on considerations such as chaotrope flux. re-
`tention of the protein and a repeatable and practical
`membrane cleaning procedure.
`In the prochymosin process, the ultrafiltration
`(U/F) feed contains about 1% suspended solids. This
`is a low enough solid content to permit use of spiral
`U/F systems which offerthe advantage of packing the
`greatest membrane surface area into the least holdup
`volume. The spiral design and resultant high mem-
`brane area to holdup volume ratio facilitates a higher
`batch concentration factor which is an important de-
`sign issue.
`
`MEMBRANE CLEANING
`
`An inclusion 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 ft2 system) with at least three holdup
`volumes (a total of about 180 gallons) of pH 10.4 di-
`lution buffer. This flush is 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 much of the product
`at nearly instantaneous dilution rates to achieve the
`best renaturation yield possible. Aslow dilution caus-
`es extreme and rapid loss of enzyme activity. 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 nextflush is made with hot
`50-54°C deionized water (D|V\I) to the waste treat-
`ment facility. 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 tank is
`filled with 50-54°C DIW. Recirculation ofthis 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 system is 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.
`Once the pH and temperature have stabilized, bleach
`is added until a free chlorine level of 180-200 ppm is
`reached. Typical bleach consumption is estimated at
`20 gallons of 5% bleach for each cleaning of a 1900
`Ft? 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 consumed as it oxidizes the gel
`layer on the membrane surface and the free chlorine
`level will drop. The pH will also drop during the first
`few bleach additions. When the pH does not drop dur-
`ing the bleach addition, it is a good indicator that the
`membrane is nearly clean. The liquid in the CIP tank
`will become clearer (turbidity will decrease) when the
`membranes are 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 50-550. The bleach is recirculated for a mini-
`mum of 20 minutes and a maximum of 60 minutes.
`
`Membrane life is adversely effected by free chlorine
`levels above 200ppm and pH 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
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`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 then filled
`with ambient temperature DIW and the system recir-
`culated. This cools the system down prior to filling
`with the next batch of feed stock and allows for the
`
`checking ofthe DIW flux which is used to benchmark
`the effectiveness of 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 possiblejust priortofilling
`the system with feed, since the membrane must be
`kept wet and the water balance on the urea recycle
`process constrains the amount of permeate that can
`be recycled.
`
`EXAMPLE 1
`
`A. PRODUCTION OF PROCHYMOSIN
`INCLUSION BODIES
`
`The preparation of prochymosin from genetically
`modified bacteria is well known in the art. See, for ex-
`ample International Patent WO 83/04418, and United
`States Patent No. 4,977,248.
`
`Briefly, is grown in 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 (|B’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
`aggregates are 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 was stirred for 90 minutes or until all
`lB’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 I 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/Abcor pilot plant U/F unit thatwas connected to
`the discharge line ofthe IB dissolution tank. The pilot
`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 made to 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 be used in this application with concentra-
`tion factors as high as 3.3X.
`
`D. DISSOLUTION RECYCLE
`
`To 3343 gms of recycle permeate that has a den-
`sity of about 1.116 and a urea concentration of about
`7.3M was added 5.8 gms ofsolid disodium phosphate
`followed by 1100 gms of 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 way to control the urea concen-
`tration from batch to batch. To 1793 gms of undiluted
`|B’s was added the 10.4 M liquid urea batch prepared
`above. The mixture was stirred at 20°C for 90 minutes
`until all |B’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 generates a dissolution with a volume
`ofabout 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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`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 amount of 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
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`Q) Om
`
`European Patent
`
`EUR
`
`EAN
`
`RCH
`
`A O T
`
`R
`
`cmegory
`A
`
`A
`
`DOCUMENTS CONSIDERED TO BE RELEVANT
`Citation of dofimgluggon, where appropriate,
`DD-A-146 751 (VEB STICKSTOFFVIERKE
`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
`
`zmt
`
`EP 94 30 7839
`
`Application Number
`
`C07K1/113
`C12N9/64
`B01D61/14
`C07B63/00
`
`
`
`TECHNICAL FIELDS
`SEARCI-[I-ID
`(lnl.CI.6)
`
`C07K
`(2078
`C 12N
`B010
`
`Page 7
`
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`
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`CATEGORY OF CITED DOCUMENTS
`
`X : particularly relevant if taken alone
`Y : particularly relevant if combined with another
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`A : technological background
`0 : non-vlrittm disclosure
`P : intermediate document
`
`Hermann, R
`1995
`24 January
`T : theory In principle underlying the invention
`E : earlier patent document, but published on. or
`aftu the filing date
`D : document cited in the application
`L : doculnult cited for other rasuns
`......................................................................................................
`& 2 munber of the same patent family. correspanding
`document
`
`_
`
`S E
`
`5
`g
`E
`2
`g
`u
`III
`E