(12) United States Patent
`Strittmatter et al.
`
`111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US006410270Bl
`US 6,410,270 Bl
`Jun.25,2002
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) PROCESS FOR THE PREPARATION OF
`RECOMBINANT PROTEINS IN E. COLI BY
`HIGH CELL DENSITY FERMENTATION
`
`FOREIGN PATENT DOCUMENTS
`
`wo
`
`9215683
`
`9/1992
`
`(75)
`
`Inventors: Wolfgang Strittmatter, Ober-Ramstadt;
`Siegfried Matzku, Zwingenberg; Dieter
`Riesenberg, Jena; Uwe Horn, Bad
`Frankenhausen; Uwe Kniipeer, Jena;
`Marian Kujau, Jena; Rolf Wenderoth,
`Jena, all of (DE); Andreas Pliickthun,
`Zurich; Anke Krebber, Zurich, both of
`(CH)
`
`(73) Assignee: Merck Patent Gesellschaft, Darmstadt
`(DE)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.:
`
`09/077,549
`
`(22) PCT Filed:
`
`Nov. 28, 1996
`
`(86) PCTNo.:
`
`PCT/EP96/05260
`
`§ 371 (c)(l),
`(2), ( 4) Date: Nov. 16, 1998
`
`(87) PCT Pub. No.: W097/21829
`
`PCT Pub. Date: Jun. 19, 1997
`
`(30)
`
`Foreign Application Priority Data
`
`Dec. 11, 1995
`
`(EP) ............................................ 95119478
`
`(51)
`
`Int. Cl? ........................ C12N 15/00; C12N 15/63;
`C12N 1!21; C12P 21/00; C07H 21/04
`(52) U.S. Cl. .................. 435/69.6; 435/69.1; 435/320.1;
`435/252.33; 435/252.3; 436/548; 536/23.1;
`536/23.533
`(58) Field of Search ............................. 435/69.1, 320.1,
`435/252.3, 252.33, 69.6, 41, 91.4, 471;
`436/548; 536/23.1, 23.53
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`OTHER PUBLICATIONS
`
`BIO/Technology, vol. 11, No. 11, Nov. 1, 1993, pp.
`1271-1277, XP000608190, P. Pack, et al., "Improved biva(cid:173)
`lent miniantibodies, with identical avidity as whole antibod(cid:173)
`ies, produced by high cell density fermentation of escheri(cid:173)
`chia coli".
`Journal of Biotechnology, vol. 39, No. 1, Feb. 21, 1995, pp.
`59-65, XP002026053, D. Korz, et al., "Simple fed-batch
`technique for high cell density cultivation of Escherichia
`coli".
`Journal of Biotechnology, vol. 32, No.3, Feb. 28, 1994, pp.
`289-298, XP002026054, K. Hellmuth, "Effect of growth
`rate on stability and gene expression of recombinant plas(cid:173)
`mids during continuous and high cell density cultivation of
`Escherichia coli TGl".
`BIO/Technology, vol. 6, Dec. 1988, pp. 1402-1405,
`XP002026055, K. Gerdes. "The PARE (HOK/SOK) locus
`of plasmid Rl: a general purpose plasmid stabilization
`system".
`
`Primary Examiner-David Guzu
`Assistant Examiner--Gerald G. Leffers, Jr.
`(74) Attorney, Agent, or Firm-Millen, White, Zelano &
`Branigan, P.C.
`
`(57)
`
`ABSTRACT
`
`The invention relates to a fed-batch fermentation process
`which uses special E. coli host/vector systems for the
`purpose of efficiently forming recombinant proteins, in
`particular recombinant antibody molecules, preferably anti(cid:173)
`body fragments such as miniantibodies. Under the given
`conditions, the E. coli cells are able to grow at a maximum
`specific growth rate up to very high cell densities. After the
`recombinant product formation has been switched on, it is
`only the formed product which restricts growth; there is no
`growth restriction due to substrates or metabolic
`by-products. High space-time yields of recombinant proteins
`can be achieved in this manner.
`
`5,558,864 A
`
`9/1996 Bendig et a!.
`
`........... 424/133.1
`
`30 Claims, 4 Drawing Sheets
`
`BEQ 1013
`Page 1
`
`

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`
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`
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`
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`
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`
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`
`02
`(B. Braun)
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`
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`
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`FIG. I
`
`--------------
`p02
`pH
`Pressure
`Temperature Revolution number
`
`Glucose
`
`Antifoam
`Weight
`
`Standard controls I
`
`Data visualization
`Process control
`
`-
`
`DCU
`
`1--
`
`Data storage
`
`Process management
`
`Date visualization
`
`Process computer
`PC 386 -MFCS
`
`Page 2
`BEQ 1013
`
`

`
`U.S. Patent
`
`Jun.25,2002
`
`Sheet 2 of 4
`
`US 6,410,270 Bl
`
`pBR322ori
`
`loci
`
`bla
`
`pHKK
`
`tHP.
`lacP/0
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`peiB
`
`VH
`
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`
`VL
`
`FIG. 2
`
`BEQ 1013
`Page 3
`
`

`
`U.S. Patent
`
`Jun.25,2002
`
`Sheet 3 of 4
`
`US 6,410,270 Bl
`
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`
`12
`
`batch
`
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`® p02
`
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`36
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`time [h]
`fed-batch
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`
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`
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`
`FIG. 3b
`
`BEQ 1013
`Page 4
`
`

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`--p02 concentration [%]
`I 111 1!11 1 1 !111 1l11 1 1 l11 1 1 !111 1l11 1 1 I
`35
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`--Oxygen concentration in the exhaust gas [%]
`I 111 1l11 1 1 l11 1 1l11 1 1 l111 1 l11 1 1l11 1 1 I
`5
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`
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`---Carbon dioxide concentration in th\
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`Page 5
`BEQ 1013
`
`

`
`US 6,410,270 Bl
`
`1
`PROCESS FOR THE PREPARATION OF
`RECOMBINANT PROTEINS IN E. COLI BY
`HIGH CELL DENSITY FERMENTATION
`
`The invention relates to a fed-batch fermentation process
`which uses special E. coli host/vector systems for the
`efficient formation of recombinant proteins, in particular
`recombinant antibody molecules, in particular antibody
`fragments such as miniantibodies.
`Under the conditions according to the invention, the E.
`coli cells can grow up to very high cell densities at a
`maximum specific growth rate. After recombinant formation
`of the product has been switched on, it is only the formed
`product which has a limiting effect on growth; substrates or
`metabolic by-products do not limit growth. In this way, and
`in conjunction with the novel expression vectors which are
`specially adapted for this purpose and which exhibit high
`stability, it is possible to achieve high space-time yields of
`recombinant proteins, which proteins exhibit high biological
`activity, in particular in the case of antibody fragments.
`The culture of E. coli cells to high cell densities is an
`essential prerequisite for efficient recombinant protein for(cid:173)
`mation. The following cultures are state of the art for this
`purpose: following unlimited growth (Jt=fAmax) in the batch
`phase, a carbon source (glucose or glycerol) is customarily
`metered in, in the subsequent fed-batch phase, in a limited
`manner such that the formation of growth-inhibiting
`by-products, for example acetate, is avoided, with the con(cid:173)
`sequence that the growth can be continued in a manner
`which is only substrate-limited (Jt<tAmaJ until high cell
`densities are reached (e.g. Riesenberg et al., 1991, J.
`Biotechnol., vol. 20, 17-28; Strandberg et al., 1994, FEMS
`Microbial. Rev., vol. 14, 53-56; Korz et al., 1995, J.
`Biotechnol. 39, 59-65; EP-B-0 511 226). Growth at a
`reduced growth rate naturally results in long fermentation
`times and consequently also in lower space-time yields.
`Owing to the immediate consumption, the concentration of
`the carbon source in the culture solution in these fermenta(cid:173)
`tions is virtually zero. The substrate-limited conditions are
`not altered after the recombinant product formation has been
`switched on.
`Fed-batch cultures which use E. coli are also known in
`which the carbon source is added discontinuously at rela(cid:173)
`tively large time intervals and then in relatively large
`quantities, with a rise in the p02 value usually being used as
`an indicator of substrate exhaustion for the purpose of
`initiating the subsequent dose of the carbon source (e.g.
`Eppstein et al., 1989, Biotechnol. 7, 1178-1181). This
`procedure means frequent switching from relatively long(cid:173)
`term substrate excess conditions to substrate limiting con(cid:173)
`ditions and consequently implies metabolic imbalances.
`In that which follows, fed-batch cultures are dealt with in
`which the cells can grow at maximum specific growth rate
`(Jt=jAmax) in the fed-batch phase. Fed-batch cultures in
`which relatively large quantities of carbon source are added 55
`to the culture at relative! y large time intervals, in accordance
`with off-line determinations, for the purpose of avoiding
`substrate limitations are experimentally elaborate and suffer
`from the disadvantage that the concentration of the carbon
`source is constantly changing during the whole of the 60
`fermentation (e.g. Pack et al., 1993, Biotechnol., vol. 11,
`1271-1277, Hahm et al., 1994,Appl. Microbial. Biotechnol.
`42, 100--107).
`Fed-batch cultures have also been described in which the
`concentration of the carbon source is measured on-line and
`is regulated so that limitations are avoided, although these
`cultures- in particular in the high cell density region-
`
`2
`suffer from the disadvantages which are described below. An
`autoclavable glucose biosensor for use of [sic] microbial
`fermentations in stirred tank fermenters has recently been
`described (M. R. Phelps et al., 1995, Biotechnol. Bioeng.,
`5 vol. 46, 514--524). It was employed for E. coli cultures. This
`in-situ sensor provides, with a time delay of approximately
`2 minutes, the current value in the culture solution. The
`signal supplied by the glucose sensor is dependent, inter alia,
`on the pH and p02 . The sensor has not been tested in the high
`10 cell density region (X>80 g/1). It is known from experience
`that growths on in-situ probes when E. coli is used can lead
`to additional erroneous values at very high cell densities. In
`addition, it is not possible to recalibrate the sensor exactly
`during an ongoing fermentation. Instead of being based on
`15 measurements using an in-situ sensor, other processes are
`based, for example, on determining the carbon sources using
`on-line flow injection analysers (FIA) or on-line HPLC in a
`culture solution which is removed semi-continuously from
`the fermenter and rendered cell-free by being subjected to
`20 filtration or microcentrifugation (Kleman et al., 1991, Appl.
`Environ. Microbial. 57, 910-917 and 918-923; Turner et al.,
`1994, Biotechnol. Bioeng. 44, 819-829). Prediction and
`feedback control algorithms have decreased the fluctuations
`in the glucose concentration during growth up to X=65 g/1
`25 (Kleman et al., 1991, Appl. Environ. Microbial., vol. 57,
`910-917). In the region of very high cell densities (from
`approx. 80 g/1 to 150 g/1 ), it becomes increasingly more
`difficult and more time consuming to separate the cells and
`the nutrient solution, so that the time delay in determining
`30 the current glucose value in the fermenter also increases in
`a biomass-dependent manner and makes it more difficult, or
`impossible, to maintain the glucose level constant. By
`contrast, the glucose concentration is measured with a time
`delay which is constant and brief using an appliance which
`35 does without this cell separation (Pfaff et al., 1995, pp. 6-11,
`in: Proceedings of the 6th International Conference on
`Computer Appl. in Biotechnol. Garmisch-Partenkirchen,
`FRG). According to the method of Pfaff et al., an FIA
`possessing an enzymic-amperometric glucose sensor is
`40 employed in the immediate vicinity of the sampling site after
`the culture has been diluted with a growth inhibitor.
`During aerobic culture, E. coli cells which are not
`obliged by the dosage regime to grow in a sub-strate-limited
`manner customarily form the metabolic by-product acetate
`45 to an increased extent (Riesenberg 1991, Curr. Opinion
`Biotechnol., vol. 2, 380--384), which acetate accumulates in
`the nutrient solution and has a growth-inhibitory effect when
`present in relatively large quantities (Pan et al. 1987, Bio(cid:173)
`technol. Lett., vol. 2, 89-94). For this reason, it has only
`50 previously been possible to effect fed-batch cultures to high
`cell densities using special E. coli strains whose accumula(cid:173)
`tion of acetate has been reduced by means of specific genetic
`alterations, while tolerating other disadvantages which are
`associated with this. The descendants of E. coli K12 include
`phosphotransacetylase-negative mutants (Bauer et al., 1990,
`Appl. Environ. Microbial., vol. 56, 1296-1302; Hahm et al.,
`1994, Appl. Microbial. Biotechnol., vol. 42, 100-107)
`whose growth is, however, strongly reduced in glucose/
`mineral salt media. Phelps and collaborators (see above)
`used the E. coli strain TOPPS as the host for non-substrate(cid:173)
`limited culture up to a biomass of X=85 g/1. However, this
`E. coli strain, which evidently does not accumulate acetate
`in a pronounced manner, is not a K12 strain. E. coli TOPPS
`forms haemolysin and is consequently a pathogenic strain
`65 which is not suitable, for reasons of safety, for use as a host
`for forming recombinant DNA products in the industrial
`sector. A reduction in acetate accumulation by means of
`
`BEQ 1013
`Page 6
`
`

`
`US 6,410,270 Bl
`
`3
`specifically reorienting the intermediary metabolism was
`achieved by transforming E. coli cells with a plasmid
`containing a gene encoding acetolactate synthase (ALS)
`(San et al., 1994, in: Ann.N.Y.Acad.Sci., vol. 721, 257-267).
`However, this procedure suffers from the disadvantage that
`instabilities usually occur under high cell density conditions
`when an ALS-encoding plasmid is used in combination with
`a second plasmid carrying the "production" gene. The
`efficiency of recombinant product formations is frequently
`decreased by plasmid instabilities, which occur to an
`increased extent particularly in association with culture to
`very high cell densities.
`Antibodies or antibody fragments, such as Fab', F(ab')2,
`miniantibodies or single-chain Fv's, are gaining ever
`increasing importance in the medical and biotechnological
`spheres. In that which follows, a miniantibody is to be
`understood, according to the invention, to be a bivalent or
`bispecific single-chain Fv fragment which is linked by way
`of a pseudo hinge region. In this context, it can be important,
`for example in cancer therapy, to make available large
`quantities of antibodies (approximately 1 g/dose ). In this
`respect, monovalent antibody fragments or fusion proteins
`of these fragments, or multimeric or multispecific variants
`thereof, can be particularly readily and satisfactorily pre(cid:173)
`pared in E. coli . These fragments or variants are of a small
`size which is associated with a high specific binding capac(cid:173)
`ity. (E.g. Pluckthun A, 1992, Immunol. Rev. 130, 151-188;
`Pack et al., 1995, J. Mol. Biol. 246, 28-34.) However,
`proteins and antibodies, in particular, must be correctly
`folded in order to be biologically and functionally active.
`When considering the yield of formed antibody fragment per
`cell, attention must be paid to this problem in connection
`with the cell density. Furthermore, the primary sequence of
`the antibody is of importance in determining the yield in
`vitro and the folding in vivo (Knappik A and Pluckthun A,
`1995, Protein Engin. 8, 81-89). Thus, Fab fragments, for
`example, are expressed as insoluble cytoplasmic or peri plas(cid:173)
`mic aggregates and refolded in vitro. Thus, yields of about
`0.14 g/1 at low cell density (Condra et al., 1990, J. Biol.
`Chern. 265, 2292-2295) and of up to about 1-2 g/1 of
`insoluble antibodies at medium cell density (Shibui et al.,
`1993, Appl. Microbial. Biotechnol. 38, 770--775) have been
`reported. Bivalent miniantibodies (Pack et al., 1993, Bio(cid:173)
`technol. 11, 1993, 1271-1277) can also be obtained in E.
`coli in biologically functional form in yields of about 0.2 g/1.
`On average, approximately 5-45% of these yields is prop(cid:173)
`erly refolded.
`In the known E. coli systems, the formation of foreign
`protein is, as a rule, switched on in a suitable manner, after
`appropriate cell densities have been reached, by a regulat(cid:173)
`able promoter system corresponding to the expression sys(cid:173)
`tem. Examples of promoter systems which may be men(cid:173)
`tioned here are (i) the araBAD promoter in the presence of
`the AraC repressor (inducible by arabinose) (e.g. Better et
`al., 1993, Proc. Natl. Acad. Sci. (USA) 90, 457-461), (ii) the
`phoA promoter (inducible by withdrawing phosphate) (e.g.
`Carteret al., 1992, Biotechnol. 10, 163-167) and (iii) the lac
`promoter system (inducible by IPTG) (Packet al., 1993, loc.
`cit.) . While the lac system brings about good expression as
`a rule, it suffers from the disadvantage that, on the one hand,
`undesirable basal expression is observed prior to induction
`of the promoter and, on the other, plasmid instability is
`observed following induction with ITPG [sic].
`In a particular embodiment of the invention, a special
`vector (pHKK) is described which contains, as the foreign
`gene, sequences which encode fragments of the murine or
`humanized antibody Mab 425. Mab 425 (ATCC HB 9629)
`
`5
`
`4
`is a murine monoclonal antibody which was isolated from
`the known human A432 cancer cell line (ATCC CRL 1555)
`and binds to the epitope of human epidermal growth factor
`receptor (EGFR, a glycoprotein of about 170 kD) while
`inhibiting the binding of the natural ligand EGF. It has been
`demonstrated that Mab 425 has a cytotoxic effect on tumour
`cells or is able to impair the growth of these cells (Rodeck
`et al., Cancer Res. 1987, 47: 3692). WO 92/15683 discloses
`humanized and chimeric forms of Mab 425, including the
`10 DNA and amino acid sequences of their light and heavy
`chains.
`The object of the invention was to make available a
`process for preparing foreign proteins, in particular antibody
`fragments, in recombinant E. coli cells under high cell
`15 density conditions (HCDC=high cell density culture) with
`high space-time yields, and without any substantial impair(cid:173)
`ment of growth by substrates or metabolites and without
`significant plasmid losses or plasmid instabilities, while
`ensuring that the expressed protein exhibits a high degree of
`20 effective biological activity (binding capacity and correct
`folding).
`The process according to the invention is a multi-step
`batch process which is primarily notable for the fact that the
`cells are able to grow at a maximum rate during the whole
`25 of the batch (jt=fAmax)· Thus, cell densities of from 100 to 150
`g/1 (bio dry mass [sic]) can ultimately be achieved using the
`described process. Furthermore, the growth is not inhibited
`to an important extent by acetate accumulation since,
`surprisingly, such an accumulation is not particularly pro-
`3D nounced under the conditions which are selected, in particu(cid:173)
`lar when E. coli strains are used which in any case only tend
`to form decreased quantities of acetate during the fermen(cid:173)
`tation. This is achieved by, also in association with a series
`of other additional measures, first and foremost, in the
`35 fed-batch phase which is inserted after a batch phase,
`keeping the concentration of the carbon source in the
`medium constant in a defined range while maintaining
`unlimited growth of the cells. By designing the relevant
`expression vector in an appropriate manner, the undesirable
`40 basal expression of protein, prior to switching on protein
`synthesis by means of a regulatable promoter system, can
`also be virtually eliminated, as can the plasmid loss, which
`is sometimes substantial and which, as already mentioned
`above, can normally be observed in expression systems
`45 using strong promoters such as the lac promoter system.
`Protein yields of on average from 3 to 5 g/1 can be
`achieved after a total culture time of from approx. 25 to 35
`hours. In the case of the antibody fragments, in particular
`miniantibodies, which are particularly critical owing to their
`50 folding criteria, approximately 80% of the synthesized mate(cid:173)
`rial is biologically active and correctly folded.
`The invention consequently relates to a process for
`preparing foreign protein, in E. coli cells which have been
`transformed with a plasmid carrying the foreign gene and an
`55 inducible promoter, by means of high cell density fermen(cid:173)
`tation by way of batch and fed-batch stages, without any
`restriction of growth by sub-strates or metabolic
`by-products, and isolation and purification of the expressed
`protein from the culture medium, with the concentration of
`60 substrates in the fed-batch phase being controlled using a
`continuous, automated or semi-automated analysis and addi(cid:173)
`tion system, with, in the fed-batch phase, (i) the concentra(cid:173)
`tion of the carbon source in the medium being kept constant
`in a range between 0.1 g/1 and 25 g/1 while maintaining
`65 unlimited growth of the cells (jt=fAmax), (ii) the production of
`the foreign protein being started in the said fed-batch phase
`by inducing the promoter at a cell density of between 10 and
`
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`US 6,410,270 Bl
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`5
`80 g/1, and (iii) utilizable nitrogen and phosphate, and also
`salts of trace elements, being fed in continuously after
`induction of product synthesis has taken place, where (iv)
`the p02 value is adjusted to between 5 and 25% during the
`whole of the fed-batch phase by passing oxygen into the 5
`fermentation broth in an appropriate manner.
`The values according to the invention for the requisite
`concentration of the carbon source during the fed-batch
`phase are in a range from 0.1 g to 25 g/1. A preferred range
`is between 0.5 and 15 g/1, in particular from 1.0 to 5 g/1, or
`from 1.0 to 3 g/1. The particularly preferred concentration is 10
`1.5 g/1. Preferred carbon sources which may be mentioned
`are glucose or glycerol or mixtures of these two compounds.
`According to the invention, the carbon source is added in a
`continuous manner (on-line) using an automated or semi(cid:173)
`automated addition and analysis system. An on-line flow 15
`injection analysis system (FIA) is preferably employed.
`The feeding-in of utilizable nitrogen, preferably ammo(cid:173)
`nium nitrogen and phosphate, for example diammonium
`hydrogen phosphate or ammonium dihydrogen phosphate,
`and also trace elements, for example salts of boron,
`manganese, copper, molybdenum, cobalt, iron or zinc which 20
`are soluble in the medium, takes place in the fed-batch phase
`which is inserted after the batch phase, preferably after
`switching on protein synthesis using the regulatable
`promoter, at a cell density of from 50 to 80 gil (bio dry mass
`[sic]), preferably at about 70 g/1, at a total growth rate [sic] 25
`of 100 to 150, preferably 140, g/1.
`According to the invention, protein synthesis is switched
`on, by activating the regulatable promoter system, at a cell
`density of from 10 to 80 g/1, preferably from 20 to 60 g/1;
`very particularly preferably, the range is from 40 to 50 g/1.
`During the fed-batch phase, the partial pressure of oxy- 30
`gen is between 5 and 25%, preferably between 15 and 25%,
`very particularly preferably 20%.
`According to the invention, the pH of the fermentation
`medium has to be adjusted, during the whole batch, to
`between 6.5 and 7.0, preferably to between 6.7 and 6.9, in 35
`particular to 6.8.
`The invention furthermore relates to a corresponding
`process in which an expression vector is employed which
`possesses an expression cassette which contained [sic] the
`foreign gene and is flanked by two terminator sequences.
`These terminator sequences, in particular that which is 40
`positioned upstream, successfully prevent unwanted expres(cid:173)
`sion of protein prior to the expression being switched on by
`the promoter system. While the terminator thp (Nohno et al.,
`1988, J. Bacterial. 170, 4097-4102) is particularly suitable,
`other known terminator sequences may also be employed. 45
`The invention furthermore relates to a process in which
`the expression vector which is employed additionally con(cid:173)
`tains a suicide system. The suicide system produces a
`protein which is toxic for the cell if the plasmid is not present
`in the cell. Suitable suicide systems are known from the 50
`literature. A suicide system which is particularly suitable for
`the invention is the hok-sok system (e.g. Gerdes K., 1988,
`Biotechnol. 6, 1402-1405). Thus, it is important, for the
`process for effectively forming recombinant proteins, in
`particular antibody molecules, that the host/vector system is 55
`characterized, in the high cell density region, by high
`plasmid stability, low recombinant basal expression and
`high product formation. In this context, suicide systems, in
`combination with recombinant expression cassettes which
`are flanked by terminators, are vector-specific.
`The invention furthermore relates to a corresponding 60
`process in which a foreign gene is employed which encodes
`an antibody fragment, in particular a miniantibody.
`The invention also relates to a process in which expres(cid:173)
`sion vectors are employed which possess additional features
`which are described below.
`In principle, most of the E. coli strains can be employed
`which are known and which are suitable for recombination
`
`6
`technology and for production on an industrial scale.
`Advantageously, those strains are preferably used which
`accumulate relatively little acetate during growth to high cell
`densities. Those strains are particularly suitable which
`exhibit an acetate enrichment of less than 5 g/1. Surprisingly,
`the acetate accumulation can be kept particularly low by
`using the chosen conditions of the process according to the
`invention. The well known and commercially available E.
`coli strain RV308 (ACCC [sic] 31608), and its variants
`having the same effect, is particularly suitable in this regard.
`The invention therefore relates, in particular, to a corre(cid:173)
`sponding process in which an E. coli strain is employed
`which exhibits an acetate accumulation of less than 5 g/1 in
`the culture medium during the fermentation.
`The invention also relates to an E. coli expression vector
`which is suitable for expressing foreign proteins under high
`cell density fermentation conditions and which exhibits the
`following features:
`
`(i)
`
`(ii)
`(iii)
`(iv)
`(v)
`
`an upstream terminator sequence and a down(cid:173)
`stream terminator sequence,
`the lac promoter/operator system,
`a T7g10 Shine Delgarno sequence,
`the pelB or ompA signal sequence,
`the sequence of the foreign gene,
`
`and, in a preferred embodiment, also a suicide system, in
`particular the hok-sok suicide system.
`According to the invention, the promoter system can also
`be replaced by other suitable systems, for example those
`mentioned above. Likewise, other signal sequences and
`control sequences having the same effect are also encom(cid:173)
`passed by the invention.
`Finally, the invention relates to the expression vector
`pHKK (FIG. 2), which is defined by its construction and
`which contains the sequences for the miniantibody which is
`derived from Mab 425, and to a special recombinant E. coli
`host RV308[pHKK], as special embodiments.
`
`DESCRIPTION OF THE FIGURES
`
`FIG. 1: Experimental set-up of the bioreactor for prepar(cid:173)
`ing proteins under high cell density conditions. The system
`is equipped with a measuring device, a display device, a
`control device and a metering device.
`FIG. 2: Optimized expression vector PHKK and constitu-
`ent parts of its construction. The vector is essentially made
`up of constituent parts from the known vectors pASK40,
`pAK100 and pKG1022.
`FIG. 3(Panels A-D): HCD culture of recombinant E. coli
`using the example of E. coli RV308[pHKK]: chronological
`course of biomass, glucose, ammonium nitrogen, phosphate,
`acetate, stirrer speed, P02 , 0 2 and C02 in the exhaust gas,
`plasmid stability (expressed as % of ~-lactamase-positive
`colonies) and formation of protein (in this case:
`scFv 425dhlx). The batch and fed-batch phases are divided
`into 5 sub-phases. The IPTG arrow indicates the start of
`protein production.
`The process according to the invention uses transformed
`E. coli host cells. The plasmid constructs which are chosen
`depend on the nature of the protein which is to be expressed.
`Features of those plasmid constructs which are particularly
`favourable are described below. The techniques and methods
`which are required for plasmid construction and for host cell
`transformation are all known and described in detail in the
`literature (e.g. Sambrook et al., 1989, Molecular Cloning: A
`65 Laboratory Manual, Cold Spring Harbor). In addition, they
`are explained in the examples using the particular embodi(cid:173)
`ments of the invention. Starting plasmids or plasmid parts
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`US 6,410,270 Bl
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`8
`which is used, this switching-on is as a rule effected by
`adding a substance or by altering a physical quantity. In the
`case of the preferred lac system (promoter, operator and
`inducer) the switching-on is effected by adding IPTG
`s (isopropyl thiogalactopyranoside ). The further growth of the
`cells is now only restricted by the accumulating product. For
`this reason, it is important, according to the invention, that
`no significant basal expression, which would exert an
`unfavourable influence on total growth and consequently on
`10 total yield, can take place prior to induction. According to
`the invention, this is contrived by the expression cassette in
`the plasmid being flanked by efficient terminator sequences.
`
`7
`are either obtainable commercially or can be constructed
`without difficulty using standard methods which are based
`on known construction schemes.
`The preceding batch phase of a typical fermentation,
`according to the invention, of transformed E. coli cells is
`divided into two subphases. Following inoculation with an
`appropriate preliminary culture, subphase I is characterized
`by a lag phase in which the cells adapt and the growth rate
`/A subsequently rises to !Amax· During subphase II, the cells
`grow exponentially at jj,=fAmax· After the p0 2 has dropped
`from 100% saturation down to less than 5-15%, the p02
`value is adjusted, by controlling the speed of the p02
`agitator, to p02 values which are preferably between 15 and
`25%, preferably around 20% (FIG. 3c). This adjustment (by
`passing in air which is enriched with pure oxygen) should be
`performed at about 6 to 12 hours after beginning fermenta(cid:173)
`tion of the main culture. The glucose concentration, which
`was preferably initially between 20 and 35 g/1, declines to
`the end of subphase II, which also constitutes the end of the
`batch phase preceding the fed-batch phase. In this
`connection, the glucose concentration values should under 20
`no circumstances fall below 0.1 g/1. From now on, this is
`prevented by the appropriate feeding-in of glucose
`(subphase III, FIG. 3a, start of the fed-batch phase). In
`accordance with the invention, the glucose value is kept
`constant between 0.1 and 25 g/1, preferably, however,