(12) United States Patent
`Andersen et al.
`
`111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US006716602B2
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6, 716,602 B2
`Apr. 6, 2004
`
`(54) METABOLIC RATE SHIFTS IN
`FERMENTATIONS EXPRESSING
`RECOMBINANT PROTEINS
`
`(75)
`
`Inventors: Dana Andersen, Redwood City, CA
`(US); John Joly, San Mateo, CA (US);
`Bradley R. Snedecor, Portola Valley,
`CA(US)
`
`(73) Assignee: Genentech, Inc., South San Francisco,
`CA(US)
`
`( *) 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.: 10/000,655
`
`(22) Filed:
`
`Nov. 1, 2001
`
`( 65)
`
`Prior Publication Data
`
`US 2002/0164700 A1 Nov. 7, 2002
`
`Related U.S. Application Data
`(60) Provisional application No. 60/245,962, filed on Nov. 3,
`2000.
`
`(51)
`
`Int. Cl? ................................................. C12P 21/06
`
`(52) U.S. Cl. .................... 435/69.1; 435/69.4; 435/71.2;
`435/252.8; 435/243
`
`(58) Field of Search ............................... 435/69.1, 69.4,
`435/71.2, 252.8, 243
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,565,785 A
`4,673,641 A
`4,710,473 A
`4,738,921 A
`4,795,706 A
`5,342,763 A
`5,612,198 A *
`5,639,635 A
`5,789,199 A *
`6,410,270 B1
`
`1!1986
`6/1987
`12/1987
`4/1988
`1!1989
`8/1994
`3/1997
`6/1997
`8/1998
`6/2002
`
`.............. 435/317
`Gilbert et a!.
`George et a!. ................ 435/68
`Morris ....................... 435/320
`Belagaje et a!. .............. 435/68
`Hsiung et a!.
`........... 435/172.3
`Schwartz ................... 435/69.1
`Brierley et a!.
`Joly et a!. .................. 435/69.7
`Joly et a!. .................. 435/69.1
`Strittmatter et a!.
`........ 435/696
`
`FOREIGN PATENT DOCUMENTS
`
`DE
`
`19943919
`
`3/2001
`
`01HER PUBLICATIONS
`
`Flamez et al., Production in Escherichia coli of a functional
`murine and murine::human chimeric F (ab')2 fragment and
`mature antibody directed against human placental alkaline
`phosphatase, 1995, Journal of Biotechnology, vol. 42, pp.
`133-143.*
`Knorre et al., High cell density fermentation of recombinant
`Escherichia coli with computer-controlled optimal growth
`rate, 1991, Ann. NY. Acad. Sci., vol. 646, pp. 300--306.*
`Skerra et al., Use of tetracycline promoter for the tightly
`regulated production of a murine antibody fragment m
`Escherichia coli, 1994, Gene, vol. 151, pp. 131-135.*
`
`Gram et al., A novel approach for high level production of
`a recombinant human parathyroid hormone fragment in
`Escherichia coli, 1994, Bio/Technology, vol. 12, pp.
`1017-1023.*
`Cheng et al., "A Novel Feeding Strategy for Enhanced
`Plasmid Stability and Protein Production in Recombinant
`Yeast Fedbatch Fermentation." Biotechnol. Bioeng.
`56:23-31 (1997).
`Cruz et al., "Metabolic Shifts by Nutrient Manipulation in
`Continuous Cultures of BHK Cells." Biotechnol. Bioeng.
`66:104-113 (1999).
`Cruz et al., "Metabolically Optimised BHK Cell Fed-Batch
`Cultures." J. Biotechnology. 80:109-118 (2000).
`Curless et al., Biotechnol. Prog. 1990, 6:149.
`Ryan et al., Biotechnol. Prog. 1996, 12:596.
`Yoon et al., Biotechnol. Prog. 1994, 43:995.
`Villa-Komaroff, et al., Proc. Natl. Acad. Sci. USA 1978,
`75:3727-373.
`DeBoer et al., Proc. Natl. Acad. Sci. USA 1983, 80:21-25.
`Sheibani, Prep. Biochem. Biotechnol. 1999, 29:77.
`Gossen et al., Curr. Opin. Biotechnol. 1994, 5:516.
`De Vos et al., Curr. Opin. Biotechnol. 1997, 8:547.
`Chevalet et al., Biotechnol. Bioeng. 2000, 69:351.
`Schroeckh et al., J. Biotechnol. 1999, 75:241.
`Staijen et al., J. Bacterial. 1999, 181:1610.
`Newman and Fuqua, Gene 1999, 277:197.
`Liu et al., Biotechniques 1998, 24:624.
`Gallia and Khalili, Ongogene 1998, 16:1879.
`Haldimann et al., J. Bacterial. 1997, 179:5903.
`Treuner-Lange et al., J. Bacterial. 1997, 179:4501.
`San et al., Ann. NY Acad. Sci. 1994, 721:268.
`Bishai et al., J. Bacterial. 1994, 176:2914.
`Lama and Carrasco, Biochem. Biophys. Res. Commun.
`1992, 188:972.
`Nielson et al., Mol. Microbial. 1991, 5:1961.
`Alksne and Rasmussen, J. Bacterial. 1997, 179:2006.
`Everett et al., Microbiology 1995, 141:419.
`
`(List continued on next page.)
`
`Primary Examiner-David Guzo
`Assistant Examiner-Daniel M. Sullivan
`(74) Attorney, Agent, or Firm-Janet E. Hasak
`
`(57)
`
`ABSTRACT
`
`The invention provides a method for increasing product
`yield of a polypeptide of interest produced by recombinant
`host cells, where expression of the polypeptide by the
`recombinant host cells is regulated by an inducible system.
`More specifically, the method involves culturing the recom(cid:173)
`binant host cells under conditions of high metabolic and
`growth rate and then reducing the metabolic rate of the
`recombinant host cells at the time of induction of polypep(cid:173)
`tide expression. In particular, the invention provides a
`method of increasing product yield of an antibody, growth
`factor, or protease produced by a recombinant E. coli host
`cell regulated by an inducible system.
`
`39 Claims, 4 Drawing Sheets
`
`BEQ 1001
`Page 1
`
`

`
`US 6,716,602 B2
`Page 2
`
`01HER PUBLICATIONS
`
`Curless et al., "Phosphate Glass as a Phosphate Source in
`High Cell Density Escherichia coli Fermentations" Biotech(cid:173)
`no!. Prog. 12:22-25 (1996).
`Hellmuth et al, "Effects of growth rate on stablility and gene
`expression of recombinant plasmids during continuous and
`high cell density cultivation of Escherichia coli TG1" Jour(cid:173)
`nal of Biotechnology 32:289-298 (1994).
`Lee and Chang, "High Cell Density Culture of a Recom(cid:173)
`binant Escherichia coli Producing Penicillin Acylase in a
`Membrane Cell Recycle Fermentor" Biotech and Bioengin
`36:330-337 (1990).
`
`Lee, Sang Yup, "High cell-density culture of Escherichia
`coli" Tibtech 14:98-105 (Mar. 1996).
`Vallejo et al., "Renaturation and purification of bone mor(cid:173)
`phogenetic protein-2-produced as inclusion bodies in high(cid:173)
`cell density cultures of recombinant Escherichia coli "
`94:185-194 (2002).
`Wilms et al., "High-Cell Density Fermentation for Produc(cid:173)
`tion of L-N-Carbamoylase Using an Expression System
`Based on the Escherichia coli rhaBAD Promoter" Biotech
`and Bioengin 73:95-103 (2001).
`
`* cited by examiner
`
`BEQ 1001
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`

`
`U.S. Patent
`
`Apr. 6, 2004
`
`Sheet 1 of 4
`
`US 6, 716,602 B2
`
`FIG. 1
`
`1200
`
`1000
`
`_j 800
`........
`0>
`E
`92 600
`0
`(.)
`t
`~ 400
`<I:
`
`200
`
`I
`ctr Is
`4.5-2.0
`3.8-2.0
`4.5-2.8
`(target 3.5) 3.3-2.8
`3.5
`3.5-3.0 4.5-2.8
`4.0-1.6
`4.5-1.9
`
`Severity of OUR shift
`
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`

`
`U.S. Patent
`
`Apr. 6, 2004
`
`Sheet 2 of 4
`
`US 6, 716,602 B2
`
`FIG. 2
`
`6
`
`5
`
`4
`
`3
`
`2
`
`c
`E
`.......
`_j
`::::::.
`0
`E
`E
`
`0::
`:::::>
`0
`
`0
`
`0
`
`10
`
`1200
`
`I
`I.
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`l
`I
`l
`I 1
`I
`l
`I 1
`I 1
`l 1
`I
`I
`l
`
`\ I
`
`20 30 40 50 60 70
`Run time ( hrs.}
`
`80
`
`Fl G. 3
`
`_j 1000
`.......
`0'1
`E 800
`ro
`0
`(.) 600
`
`I -c:
`
`<! 400
`
`200
`
`0
`
`0
`
`5
`
`10
`
`15
`25
`20
`30
`Hours post- induction
`
`35 40
`
`BEQ 1001
`Page 4
`
`

`
`U.S. Patent
`
`Apr. 6, 2004
`
`Sheet 3 of 4
`
`US 6, 716,602 B2
`
`Fl G. 4A
`
`1200
`
`1000
`
`-_j 800
`
`.........
`Ol
`E 600
`1'--
`rn
`0 >-
`
`400
`
`200
`
`0
`2.0
`
`30
`
`40
`
`50
`Time ( hrs)
`
`I
`60
`
`I
`70
`
`I
`80
`
`FIG. 48
`
`900
`
`-_j 700
`
`500
`
`.........
`CJ'I
`E
`1'--rn
`0 300
`>-
`
`100
`
`30
`
`40
`
`50
`Time (hrs)
`
`60
`
`70
`
`BEQ 1001
`Page 5
`
`

`
`U.S. Patent
`
`Apr. 6, 2004
`
`Sheet 4 of 4
`
`US 6, 716,602 B2
`
`Fl G. 5A
`
`10
`
`8
`
`6
`
`c
`·-
`E
`........
`_J
`........
`0
`E 2
`E
`
`4
`
`0
`
`0
`
`20
`
`40
`Time (hrs)
`
`60
`
`80
`
`FIG. 58
`
`8
`
`6
`
`4
`
`2
`
`0
`
`c ·-
`E
`.........
`_J
`........
`0
`E
`E
`
`0
`
`20
`
`40
`Time (hrs}
`
`60
`
`80
`
`BEQ 1001
`Page 6
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`

`
`US 6,716,602 B2
`
`1
`METABOLIC RATE SHIFTS IN
`FERMENTATIONS EXPRESSING
`RECOMBINANT PROTEINS
`
`This application claims priority under 35 U.S.C. §119(e) 5
`from U.S. Provisional Application Serial No. 60/245,962,
`filed Nov. 3, 2000.
`
`FIELD OF THE INVENTION
`
`The present invention relates to improvements in product
`yield from fermentation to produce recombinant proteins,
`particularly in prokaryotic and simple eukaryotic systems.
`More particularly, this invention greatly increases the yield
`of properly assembled proteins in large scale fermentations.
`
`BACKGROUND OF THE INVENTION
`
`2
`between these factors result in a decrease of the overall yield
`below its theoretical potential. Consequently, some interme(cid:173)
`diate growth rates may be more favorable for the production
`of high quantities of high quality product.
`An added complication is that induction of recombinant
`protein expression essentially highjacks the cellular protein
`assembly process to make large quantities of a product with
`no benefit, and often with significant detriment, to the cell.
`In fact, for cases in which induction is triggered by phos-
`10 phate depletion using the alkaline phosphatase promoter,
`growth rate is dramatically curtailed by the phosphate star(cid:173)
`vation itself. This effect does not affect the metabolic rate,
`however.
`Thus, there is a need in the art to increase the yield of
`15 usable recombinant protein production. The present inven(cid:173)
`tion advantageously and unexpectedly addresses this need
`by permitting high levels of protein synthesis, assembly and
`folding. Because different factors may play critical roles in
`maximizing usable protein yield prior to induction during
`20 the growth phase of the culture, and post-induction, the
`independent control of these two factors can lead to
`improved yields of usable products, such as for the case of
`soluble, properly folded and assembled antibody fragments.
`
`The production of large quantities of relatively pure,
`biologically active polypeptides and proteins is important
`economically for the manufacture of human and animal
`pharmaceutical formulations, enzymes, and other speciality
`chemicals. Recombinant DNA techniques have become the
`method of choice to produce large quantities of exogenous
`proteins using bacteria and other host cells. The expression
`of proteins by recombinant DNA techniques for the produc-
`tion of cells or cell parts that function as biocatalysts is also
`an important application.
`Producing recombinant protein involves transfecting host
`cells with DNA encoding the protein and growing the cells
`under conditions favoring expression of the recombinant
`protein. The prokaryote E. coli is favored as host because it
`can be made to produce recombinant proteins in high yields.
`Numerous U.S. patents on general bacterial expression of
`DNA encoding proteins exist, including U.S. Pat. No. 4,565,
`785 on a recombinant DNA molecule comprising a bacterial 35
`gene for an extracellular or periplasmic carrier protein and
`a non-bacterial gene; U.S. Pat. No. 4,673,641 on
`co-production of a foreign polypeptide with an aggregate(cid:173)
`forming polypeptide; U.S. Pat. No. 4,738,921 on an expres(cid:173)
`sion vector with a trp promoter/operator and trp LE fusion
`with a polypeptide such as IGF-I; U.S. Pat. No. 4,795,706 on
`expression control sequences to include with a foreign
`protein; U.S. Pat. No. 4,710,473 on specific circular DNA
`plasmids such as those encoding IGF-I; U.S. Pat. No.
`5,342,763 on improving expression in bacteria by manipu(cid:173)
`lating oxygen delivery; and U.S. Pat. No. 5,639,635 on
`secretion of the expressed protein into the bacterial peri(cid:173)
`plasm.
`Recombinant proteins become less expensive if the fer(cid:173)
`mentation yield improves. Yield depends upon the rate at
`which the recombinant protein is properly folded and
`assembled protein is formed and upon the length of time
`over which the protein is produced.
`The recombinant protein expression rate is typically
`affected by the growth and metabolic rates of the cells. At 55
`higher growth rates, the rate at which a protein can be
`expressed when induced typically increases (Curless et al.,
`Biotechnol. Prog. 1990, 6:149). However, upon induction,
`high protein expression rates may not always lead to high
`rates of formation of active, properly formed products. In 60
`other words, while the quantity of protein translated may be
`maximized, other factors may compromise the quality of the
`product, such as degradation of the protein by proteases or
`other detrimental post-translational modifications (Ryan et
`al., Biotechnol. Prog. 1996, 12:596; Yoon et al., Biotechnol. 65
`Prog. 1994, 43:995). Efficient fermentation requires balanc(cid:173)
`ing growth rate against yield of usable protein; compromises
`
`25
`
`SUMMARY OF THE INVENTION
`The invention provides a method for increasing product
`yield of a polypeptide of interest produced by recombinant
`host cells, where expression of the polypeptide by the
`recombinant host cells is regulated by an inducible system.
`30 More specifically, the method involves culturing the recom(cid:173)
`binant host cells under conditions of high metabolic and
`growth rate, then reducing the metabolic rate of the recom(cid:173)
`binant host cells at the time of induction of polypeptide
`expression.
`In a specific embodiment the invention provides a method
`of increasing product yield of an antibody, growth factor, or
`protease produced by a recombinant E. coli host cell regu(cid:173)
`lated by an inducible system.
`In a further specific embodiment, the invention provides
`a method of increasing the yield of actively folded proteins
`having different structures, for example Fab'2 versus Fab Fv
`antibody fragments, by selecting the time to initiate reduc(cid:173)
`tion in metabolic rate (the rate shift), the rate of adjustment
`(shift) of the metabolic rate, and the final metabolic rate.
`Adjusting these parameters of the invention enhances the
`yield of correctly folded proteins having different secondary
`and tertiary structures, interaction and refolding
`characteristics, size and contact area, and other factors that
`can affect protein assembly and function.
`
`40
`
`45
`
`50
`
`DESCRIPTION OF THE DRAWINGS
`FIG. 1 shows anti-CD18 yield (titer) in a series of
`anti-CD18 Fab'2 fermentations. X-axis: approximate oxygen
`uptake rate which reflects the severity of the oxygen use rate
`shift.
`FIG. 2 shows actual oxygen uptake rate profile from the
`fermentations in FIG. 1. Titers exceeding 1000 mg!L are
`represented by a thin solid line (--); titers between
`800-900 mg/L are represented by a dotted line(-----); and
`the unshifted control (-600 mg/L) is represented by a heavy
`solid line (--).
`FIG. 3 shows the titer results for quantitating antibody
`production of the various runs as a function of fermentation
`time. A less severe shift is represented by squares ( ---0---),
`a more severe shift is represented by triangles(."---) and the
`control is represented by diamonds ( + ).
`
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`

`
`US 6,716,602 B2
`
`3
`FIGS. 4A and 4B show titer profiles for a series of
`anti-VEGF fermentations or without (B) oxygen use rate
`(OUR) shifts. The graphs each present data from three runs.
`Run 1 is represented by diamonds ( + ), Run 2 is represented
`by squares (•) and Run 3 is represented by triangles ( .. ).
`FIGS. SA and 5B show OUR profiles for the runs in FIG.
`4. Run 1 is represented by diamonds ( + ), Run 2 is repre(cid:173)
`sented by squares (•) and Run 3 is represented by triangles
`
`( .. ).
`
`DETAILED DESCRIPTION
`
`The present invention advantageously provides a method
`for increasing yield of a heterologous recombinant protein
`produced by recombinant host cells by first increasing the
`protein production capacity of the cells in culture by cul(cid:173)
`turing the cells at a high growth rate, and then decreasing
`metabolic rate of the cells (rate shift) to permit proper
`folding or assembly of the heterologous protein. In a specific
`embodiment, implementing a high growth rate was found to
`extend the period of heterologous gene expression. In a
`further specific embodiment, the metabolic rate shift
`increases the yield of properly folded and, if appropriate,
`assembled protein. These features together increase fermen(cid:173)
`tation efficiency.
`The invention is based on observations in a number of E.
`coli fermentations producing anti-CD18 Fab' 2 and anti(cid:173)
`VEGF Fab, that the deliberate down shifting of the cellular
`metabolic rate of the cells (by manipulating the oxygen
`transfer rate and correspondingly, the glucose feed rate in the
`fermentor) significantly improves product yields. In
`particular, growing the cells at a relatively high metabolic
`rate, and then dramatically shifting down the metabolic rate
`after the induction of antibody expression greatly improves
`yield. A substantial amount of data demonstrates that this
`approach extends the period of antibody fragment assembly,
`leading to significantly higher titers.
`These experiments also established that for any given
`heterologous protein expression system, i.e., the nature of
`the protein and characteristic of the host, tuning the meta(cid:173)
`bolic rate shift further increases useful protein yields. The
`tuning variables include the tuning of the metabolic rate
`shift, the step-down rate (rate of decrease in available
`oxygen or carbon/energy source, or both), and the final
`metabolic rate (available oxygen level, available carbon/ 45
`energy source level or both).
`Consequently, post-induction, the protein expression rate
`can be controlled by manipulating the metabolic rate, one
`common measure of which is the oxygen uptake rate of the
`cells in the fermentor. Metabolic rate control can be 50
`achieved by controlling the feeding of the primary carbon
`source, commonly glucose, often in conjunction with
`manipulation of fermentor parameters such as agitation rate
`and back pressure, to control the oxygen transfer rate to the
`cells. Conversely, metabolic rate control can be achieved by 55
`limiting the available oxygen, in conjunction with a reduc(cid:173)
`tion in the glucose feed rate. Similar trade-offs exist between
`protein synthesis rate and the rate of formation of usable
`product for controlling the metabolic rate post-induction as
`previously discussed for controlling growth rate pre- 60
`induction. For the case of maximizing the yield of antibody
`fragments, the rate and period of assembly of soluble, active
`product from the individual light-chain and heavy-chain
`polypeptides occurs at some favorable post-induction meta(cid:173)
`bolic rate.
`While data in the literature suggests that fermentations
`may have a favorable growth rate for protein production, the
`
`30
`
`4
`results in this application show that the shifting of metabolic
`rates in different phases of the fermentation provides a
`critical benefit. In other words, we see significantly
`improved product yields by shifting the metabolic rate
`5 compared to the titers obtained by running the fermentation
`at a previously favorable, constant metabolic rate. While all
`of the data to date has been obtained using fermentations
`producing antibody fragments in E. coli, this approach
`applies to a variety of proteins, including growth hormone,
`10 expressed in other prokaryotic and simple eukaryotic sys(cid:173)
`tems.
`As used herein, "reducing metabolic rate" or "shifting
`down metabolic rate" means altering the host cell culture
`conditions such that the host cells undergoing rapid growth
`15 and expansion reduce (or stop) growth and expansion. For
`the case of cells already in a reduced growth state, the rates
`of oxygen uptake and the corresponding rates of uptake of
`a carbon/energy source are reduced. Since, in the case of
`respiring cells, the metabolic rates are determined primarily
`20 by the rate at which the cell oxidizes the available carbon/
`energy source using the available oxygen, the metabolic rate
`can be reduced by limiting either of these two reactants. So
`reduction of metabolic rate can result from inter alia (1)
`reducing the amount of available oxygen in the cell culture
`25 (i.e., fermentation); (2) reducing the amount of available
`carbon/energy sources; or (3) reducing both.
`The term "available oxygen" refers to oxygen that can be
`used by the cells. "Decreasing available oxygen" can be
`effected by decreasing the oxygen transfer rate to the culture,
`or decreasing the oxygen transfer by the cells or both. Often
`it is desirable to reduce the feed rate of glucose (or altern a(cid:173)
`tive carbon/energy source) correspondingly, and so the dis(cid:173)
`solved oxygen concentration may be decreased or not,
`depending on which reactant most directly limits respiration.
`As used herein, the phrase "carbon/energy source" refers
`to a source of carbon and energy for the cells. Examples of
`such a source include glycerol, succinate, lactate, and sugars
`such as, e.g., glucose, lactose, sucrose, and fructose. The
`40 selection of the particular carbon/energy source to employ
`will depend mainly on the characteristics of the host cell.
`The preferred carbon/energy source for E. coli fermentation
`is glucose.
`Thus, decreasing available carbon/energy sources can
`mean reducing the concentration or feed rate of the carbon/
`energy source, or reducing the rate of transfer to the host
`cells or uptake by the host cells of the carbon/energy source,
`or both.
`As used herein "culturing the host cells under conditions
`of high metabolic and growth rate" means establishing the
`host cell culture conditions to favor growth. e.g., by pro(cid:173)
`viding unrestricted or relatively high feed rates of nutrients
`energy and oxygen, such that the cells have rapid growth and
`metabolic rates prior to reducing metabolic rate to increase
`"product yield". Under these conditions host cell doubling
`time decreases towards its minimum and host cell metabo-
`lism increases exponentially towards its maximum, poten(cid:173)
`tially achieving either or both conditions. Measurement of
`metabolic And growth rates is easily determined using
`routine techniques, including but not limited to measure(cid:173)
`ment of increases in cell number, measurement of increases
`in cell density (e.g., optical density), measurement of pH
`changes of the growth medium containing the cell, mea(cid:173)
`surement of accumulated metabolites, measurement of heat
`65 production, measurement of electrical conductivity of the
`medium, measurement of nutrient feed rates, and measure(cid:173)
`ment of oxygen uptake and carbon dioxide evolution rates.
`
`35
`
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`
`US 6,716,602 B2
`
`5
`As used herein, the term "product yield" refers to the
`quantity of useful recombinant protein produced by a fer(cid:173)
`mentation system. Protein quantity is readily determined
`using routine techniques, including but not limited to
`chromatography, spectrometry gel electrophoresis, 5
`immunoassay, coomassie blue or silver staining, and the
`Lorry essay. Protein quality is further evaluated by compar(cid:173)
`ing product to a standard in appropriate biophysical or
`activity assays, e.g., high performance liquid
`chromatography, spectroscopic analysis, or immunoassay. 10
`Activity assays can reveal properly folded or assembled
`functional protein. Thus, properly assembled antibody may
`bind antigen, preferably with similar affinity as a control
`antibody. A properly assembly growth factor, hormone, or
`cytokine will bind its cognate receptor and induce cell 15
`signaling, again in a manner comparable to that of wild-type
`growth factor, hormone, or cytokine. A properly refolded
`protease will cleave peptide bonds with similar specificity to
`that of a wild-type protease.
`As used herein, "polypeptide of interest" refers generally 20
`to peptides and proteins having more than about 10 amino
`acids. The polypeptides may be endogenous to the bacterial
`host cell, or, preferably, may be exogenous to the host cell,
`such as yeast polypeptides, or more preferably, mammalian
`polypeptides. Examples of bacterial polypeptides include, 25
`e.g, alkaline phosphatase and beta-lactamase. Examples of
`mammalian polypeptides include molecules such as, e.g.,
`renin, a growth hormone, including human growth hormone,
`des-N-methionyl human growth hormone, and bovine
`growth hormone; growth hormone releasing factor; parathy- 30
`raid hormone; thyroid stimulating hormone; thyroxine; lipo(cid:173)
`proteins; alphal-antitrypsin; insulin A-chain; insulin
`B-chain; proinsulin; follicle stimulating hormone; calcito(cid:173)
`nin; leutinizing hormone; glucagon; clotting factors such as
`factor VIIIC, factor IX, tissue factor and Von Willebrands 35
`factor; anti-clotting factors such as Protein C; atrial naturi(cid:173)
`etic factor; lung surfactant; a plasminogen activator, such as
`urokinase or human urine or tissue-type plasminogen acti(cid:173)
`vator (t-PA); bombesin; thrombin; hematopoietic growth
`factor; tumor necrosis factor-alpha and -beta; enkephalinase; 40
`a serum albumin such as human serum albumin; mullerian(cid:173)
`inhibiting substance; relaxin A-chain; relaxin B-chain; pro(cid:173)
`relaxin; mouse gonadotropin-associated peptide; a microbial
`protein, such as beta-lactanase; DNase; inhibin; activin;
`vascular endothelial growth factor; receptors for hormones 45
`or growth factors; integrin; thrombopoietin; protein A or D;
`rheumatoid factors; a neurotrophic factor such as bone(cid:173)
`derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5,
`or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor
`such as NGF-beta; platelet-derive growth factor (PDGF); 50
`fibroblast growth factor such as aFGF and bFGF; epidermal
`growth factor (EGF); transforming growth factor (TGF)
`such as TGF-alpha and TGF-beta, including TGF-betal,
`TGF-beta2, TGF-beta3, TGF-beta4, or TGF-beta5; insulin(cid:173)
`like growth factor-! and -II (IGF-1 and IGF-11); insulin-like 55
`growth factor binding proteins; CD proteins such as CD-3,
`CD-4, CD-8, and CD-19; erythropoietin; osteoinductive
`factors; immunotoxins; a bone morphogenetic protein
`(BMP); somatotropins; interferons such as interferon-alpha,
`-beta, and -gamma; colony stimulating factors (CSFs), e.g., 60
`M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-l
`to IL-15; superoxide dismutase; T-cell receptors; surface
`membrane proteins; decay accelerating factor; viral
`antigens, such as, for example, a portion of the AIDS
`envelope; transport proteins; homing receptors; addressins; 65
`regulatory proteins; antibodies; and fragments of any of the
`above-listed polypeptides.
`
`6
`As used herein, the term "antibody" refers to full-length
`immunoglobins (IgA, IgD, IgE, IgG, IgM) and all isotypes
`thereof, humanized or chimeric antibodies, multispecific
`antibodies, CDR-modified antibodies, and antibody frag(cid:173)
`ments thereof. Antibody fragments include Fab'2 , Fab, scFv
`single chain antibodies, and the like.
`The preferred polypeptides of interest are those that are
`easily produced in bacterial cells with a minimum of pro(cid:173)
`teolysis and a maximum in properly refolded or active
`material and need not be glycosylated for their intended
`utility. Examples of such mammalian polypeptides include
`antibodies (or fragments thereof), IGF-1, growth hormone,
`DNase, relaxin, growth hormone releasing factor, insulin,
`urokinase, immunotoxins, and antigens. Particularly pre(cid:173)
`ferred mammalian polypeptides include antibodies, IGF-1,
`and growth hormone.
`A modified "host cell" is a cell in which a nucleic acid
`encoding the polypeptide of interest has been introduced.
`Alternatively the polypeptide of interest can be encoded by
`a gene that is part of the cell's genome, but for which
`regulatory sequences have been modified to provide for
`increased levels of expression.
`Examples of host cells include, but are not limited to,
`bacterial organisms (bacteria), archaebacteria, simple single
`celled eukaryotes such as yeast and other fungi, plant cells,
`and animal cells. Suitable bacteria for this purpose include
`aerobic and facultative anaerobic bacteria, whether archae(cid:173)
`bacteria and eubacteria, especially eubacteria, and most
`preferably Enterobacteriaceae. Examples of useful bacteria
`include Escherichia, Enterobacter, Azotobacter, Erwinia,
`Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella,
`Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.
`Suitable£. coli hosts include£. coli W3110 (ATCC 27,325),
`E. coli 294 (ATCC 31, 446), E. coli B, and E. coli X1776
`(ATCC 31,537). These examples are illustrative rather than
`limiting. Mutant cells of any of the above-mentioned bac(cid:173)
`teria may also be employed. It is, of course, necessary to
`select the appropriate bacteria taking into consideration
`replicability of the replicon in the cells of a bacterium. For
`example, E. coli, Serratia, or Salmonella species can be
`suitably used as the host when well known plasmids such as
`pBR322, pBR325, pACYC177, or pKN410 are used to
`supply the replicon. E. coli strain W3110 is a particularly
`preferred parent host because it is a common host strain for
`recombinant DNA product fermentations. Preferably, the
`host cell should secrete minimal amounts of proteolytic
`enzymes. For example, strain W3110 may be modified to
`effect a genetic mutation in the genes encoding proteins,
`with examples of such hosts including E. coli W3110 strain
`1 A2, which has the complete genotype ll fhuA; E. coli
`W3110 strain 9E4, which has the complete genotype ll
`fhuA-ptr3; E. coli W3110 strain 27C7 (ATCC 55,244),
`which has the complete genotype ll fhuA-ptr3 phoA-ll-E15-
`ll-(argF-lac) 169 ompT-ll-degP41kanr; E. coli W3110 strain
`37D6, which has the complete genotype ll fhuA-ptr3 phoA(cid:173)
`ll-E15-ll-( argF -lac )169 ompT-ll-degP41kanr rbs7 -ll-ilvG;
`E. coli W3110 strain 40B 4, which is strain 37D6 with a
`non-kanamycin resistant degP deletion mutation; and an E.
`coli strain having mutant periplasmic protease disclosed in
`U.S. Pat. No. 4,946,783 issued Aug. 7, 1990. Examples of
`mammalian cells are COS-1 or CHO cells, HeLa cells, 293T
`(human kidney cells), mouse primary myoblasts, and NIH
`3T3 cells. Examples of yeast species are S. cerevisiae,
`Candida albicans, Candida utilis, and Phaffia rhodozyma.
`Other suitable host cells are insect cells such as SF -9 cells
`(Spodoptera frugiperda).
`Host cells grow under amenable culture conditions, i.e.,
`appropriate conditions of temperature (generally around
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`US 6,716,602 B2
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`7
`25-37° C.), pH (generally pH 7-8), humidity (generally
`about 100%), oxygen, and nutrient availability including
`carbon/energy sources. As described herein, availability of
`oxygen and an energy source determine host cell growth
`rate.
`As used herein, "large-scale" fermentation refers to fer(cid:173)
`mentation in a fermentor that is at least approximately 1000
`liters in volumetric capacity, i e., working volume, leaving
`adequate room for headspace. "Small-scale" fermentation
`refers generally to fermentation in a fermentor that is no
`more than approximately 100 liters in volumetric capacity,
`preferably no more than approximately 10 liters.
`
`Recombinant Host Cells
`
`5
`
`8
`or enzyme, i.e., the nucleotide sequence encodes an amino
`acid sequence for that polypeptide, protein or enzyme. A
`coding sequence for a protein may include a start codon
`(usually ATG) and a stop codon.
`The term "gene", also called a "structural gene" means a
`DNA sequence that codes for or corresponds to a particular
`sequence of amino acids which comprise all or part of one
`or more proteins or enzymes, and may or may not include
`regulatory DNA sequences, such as promoter sequences,
`10 which determine for example the conditions under which the
`gene is expressed. Some genes, which are not structural
`genes, may be transcribed from DNA to RNA, but are not
`translated into a polypeptide sequence. Other genes may
`function as regulators of structural genes or as regulators of
`15 DNA transcription.
`The terms "express" and "expression" mean allowing or
`causing the information in a gene or DNA sequence to
`become manifest, for example producing a protein by acti(cid:173)
`vating the cellular functions involved in transcription and
`translation of a corresponding gene or DNA sequence. A
`DNA sequence is expressed in or by a cell to form an
`"expression product" such as a protein. The expression
`product itself, e.g., the resulting protein, may also be said to
`be "expressed" by the cell. An expression product can be
`characterized as intracellular, extracellular or secreted. The
`term "intracellular" means something that is inside a cell.
`The term "extracellular" means something that is outside a
`cell. A substance is "secreted" by a cell if it appears in
`significant measure outside the cell, from somewhere on or
`inside the cell.
`The term "expression system" means a host cell and
`compatible vector u