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`Biotechnology Letters, Vol 19, No 11, November 1997, pp. 1079–1082
`
`Novel fed-batch strategy
`for the production of insulin-like
`growth factor 1 (IGF-1)
`N.D. Wangsa-Wirawan, Y.S. Lee, R.J. Falconer, C.J. Mansell, B.K. O’Neill
`and A.P.J. Middelberg*
`Department of Chemical Engineering, The University of Adelaide, Adelaide 5005, Australia;
`*Email: antonm@chemeng.adelaide.edu.au, Fax + 61-8-8303-4373
`
`The production of Long-R3-IGF-1 (an IGF-1 fusion analog) by constant-rate, fed-batch fermentation of Escherichia
`coli yielded 2.6 g fusion protein/L, corresponding to an actual IGF-1 concentration of 2.2 g/L. A novel strategy
`employing three distinct feeding stages was developed which raised product concentration to 4.3 g/L (3.6 g/L of
`IGF-1) while minimising glucose and acetate accumulation. This improved productivity was not accompanied by an
`increase in inclusion body size.
`
`Introduction
`Fed-batch fermentation has been commonly used to
`achieve high-cell-density cultivation. There are several
`feeding strategies that can be applied to achieve
`different goals (Yamane and Shimizu, 1984; Lee, 1996).
`Exponential feeding has been successfully applied for
`the high-cell-density culture of several non-recombinant
`and recombinant E. coli strains (Strandberg and Enfors,
`1991; Yee and Blanch, 1993; Helmulth et al., 1994)
`and leads to less acetate formation.
`
`An analog of insulin-like growth factor 1, termed
`Long-R3-IGF-1 (Francis et al., 1992), is produced as a
`solid inclusion body (IB) when expressed at high levels
`in recombinant E. coli. This protein is produced by
`GroPep Pty Ltd, Adelaide, and sold for use as a
`mammalian cell-culture
`supplement. Demand
`for
`culture media and consequently this protein is expected
`to increase as new biopharmaceutical products approach
`phase III clinical trials. Additionally, the fusion protein
`can be easily cleaved to give pure IGF-1, which has
`several clinical uses. There is consequently a need to
`improve the productivity of the current growth factor
`production process, preferably through process intensi-
`fication. A key unit to be optimised for higher yield is
`the fermenter. The existing strategy uses a constant-rate
`fed-batch system and achieves approximately 20 g cell
`dry weight (CDW)/L. The resulting inclusion body is
`small, with a typical median diameter of 0.3 mm,
`making separation of the inclusion bodies from the
`cellular debris difficult
`following high-pressure
`homogenisation.
`
`This paper details our initial attempts to increase the
`fermenter productivity and inclusion body size through
`improved control and feeding strategies. A series of
`fermentations was conducted where the feeding strategy
`was altered. A novel 3-stage feeding strategy was devel-
`oped. Oxygen transfer was also enhanced through the
`use of oxygen-enriched air feed.
`
`Materials and methods
`Strains and Growth
`E. coli strain JM101 [SupE thiD (lac- proAB) F¢[traD36
`proAB+ lacIq lacMZ M15]] containing the strictly-regu-
`lated plasmid p[Met1]-pGH(1–11)-Val-Asn-[Arg3]-
`IGF-1 (Francis et al., 1992) was grown on a modified
`C1 media agar plate (containing 100 mg Amp/L) and
`incubated at 37(cid:176)C for 24 h.
`
`Shake Flask Culture
`An inoculum for the fermenter was prepared in a shake
`flask containing 20 mL of sterile modified C1 media.
`This shake flask was inoculated with a single colony
`from the plate, then incubated at 37(cid:176)C in a shaker at
`200 rpm for 11 hours.
`
`Fed-batch Fermentation
`Fermentation was conducted in a 20-L fermenter with
`an initial volume of 12 L of modified C1 media (see
`below). A volume of inoculum was added to the
`fermenter and left overnight to give 2.4 g CDW/L at
`9 am on the following day. The pH of the culture was
`maintained at 6.9 using 25% ammonia solution.
`Temperature was 37(cid:176)C and dissolved O2 concentration
`
`© 1997 Chapman & Hall
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`Figure 1 Fed-batch fermentation profiles using constant-
`rate feeding. u, dry cell weight (DCW); r, acetate concen-
`tration; m, glucose concentration. (a) feed rate = 9.7 mL/min;
`(b) feed rate = 11.4 mL/min.
`
`accumulation following induction is also typically
`observed (Figure 1). An inclusion body size of 0.32 mm
`and a fusion-protein concentration of 2.6 g/L, corre-
`sponding to an actual IGF-1 concentration of 2.2 g/L,
`were obtained for the fermentation reported in Figure 1a.
`
`The high acetate and glucose concentrations in Figure
`1 are due to a rapid decrease in specific growth rate
`following induction (Lee and Ramirez, 1992). A feeding
`strategy employing three different stages was therefore
`designed to overcome this problem. In stage 1, a high
`specific growth rate (0.25 h–1) was used to generate a
`high cell density before induction, and hence a large
`cell population for the production of recombinant
`protein. In stage 2, a rapidly-decreasing feedrate that is
`designed to approximate the rapid drop in metabolic
`activity following IPTG induction was employed.
`Finally, the lower and variable metabolic activity
`following induction was approximated with a glucose
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`J BL,1079-1082,870 Wira 20/10/97 8:17 am Page 1080
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`N.D. Wangsa-Wirawan et al.
`
`(DOC) was greater than 60% saturation. Aeration was
`switched to oxygen-enriched air (mixture of 25% air
`and 75% oxygen) when the DOC dropped below 60%,
`at an approximate cell concentration of 3 g CDW/L.
`Nutrient feeding commenced when glucose exhaustion
`occurred, at an approximate cell concentration of 13.5 g
`CDW/L. After 4 h of feeding, the culture was induced
`isopropyl-b-D-thiogalactopyranoside
`with
`0.2 mM
`(IPTG) and the fermentation was terminated 5 h later.
`Two different
`feeding strategies were employed;
`constant-rate feeding and a novel 3-stage feeding
`strategy. For constant feeding, nutrient feed was
`pumped at a rate of 9.7 mL/min or 11.4 mL/min. 3-
`stage feeding was PLC-controlled. The initial stage
`consisted of exponential feeding for 4 h to give a
`constant specific growth rate of m= 0.25 h–1 prior to
`induction. Immediately following induction, a linearly-
`decreasing feed rate was used for 0.5 h, followed by
`exponential feeding for 4.5 h at a specific growth rate
`of m= 0.1 h–1. OD600, dry cell weight (DCW), glucose
`concentration, and acetate concentration were measured
`throughout the fermentation. Glucose concentration was
`measured using a YSI analyser (2700 SELECT, Yellow
`Springs Instruments, USA). Acetate concentration was
`determined using an enzyme assay kit obtained from
`Boehringer Mannheim (Catalog Number: 148261).
`Inclusion body size was measured using a Joyce Loebl
`Disc Centrifuge (Middelberg et al., 1990). Recombinant
`protein concentration was determined by High Perfor-
`mance Liquid Chromatography (Falconer et al., 1997).
`
`Modified C1 media
`D-Glucose.H2O, 2.96 g/L
`and
`shake flask)
`(for
`40.0 g/L (for fermenter); NH4Cl, 2.58 g/L; KH2PO4,
`2.54 g/L; Na2HPO4, 4.16 g/L; K2SO4, 1.94 g/L;
`MgSO4.7H2O,
`0.67 g/L;
`20 mg/L;
`FeSO4.7H2O,
`MnSO4.H2O, 5.0 mg/L; ZnSO4.7H2O, 8.6 mg/L;
`CuSO4.5H2O, 0.76 mg/L; trisodium citrate, 88 mg/L;
`thiamine, 48 mg/L.
`
`Nutrient feed
`D-Glucose.H2O, 620 g/L; KH2PO4, 5.3 g/L; Na2HPO4,
`79.0 g/L; K2SO4, 45.0 g/L; MgSO4.7H2O, 8.24 g/L. In
`yeast extract-supplemented nutrient feed, nutrient feed
`as above was supplemented with 65 g/L yeast extract.
`
`Results and discussion
`Fermentation profiles for the constant-rate feeding strat-
`egy are presented in Figure 1. Glucose accumulated to
`high concentrations (up to 40 g/L), and relatively low
`cell-densities were obtained. The highest OD600 previ-
`ously achieved using this standard production method
`was approximately 80 (unpublished data). High acetate
`
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`J BL,1079-1082,870 Wira 20/10/97 8:17 am Page 1081
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`Novel fed-batch strategy for the production of insulin-like growth factor 1
`
`feed rate designed to support a constant specific growth
`rate of 0.1 h–1. The feed profile is summarised in
`Figure 2. Stages 2 and 3 are an approximation to the
`changes in growth rate observed due to induction shock,
`as described by the shock-recovery model of Lee and
`Ramirez (1992).
`
`The fermentation profiles for 3-stage feeding are pre-
`sented in Figure 3a. An OD600 of 150, corresponding
`to a dry cell weight of 42 g/L, was achieved. This was
`nearly double that obtained using constant-rate feeding.
`Glucose accumulated
`in
`fermentation broth was
`decreased to 6.2 g/L, approximately 15% of the glucose
`level using constant-rate feeding. This method therefore
`overcomes glucose accumulation problems. After induc-
`tion, acetate gradually accumulated and reached a level
`of 44 mM at the end of fermentation. This acetate
`concentration is still excessive, but can be reduced by
`further optimisation of the feed profile. For example,
`the drop in metabolic activity post induction could be
`better approximated using three stages: a linearly
`decreasing stage; a stage with m = 0.1 h–1; and a stage
`with some constant m < 0.1 h–1.
`
`A recombinant protein concentration of 4.3 g/L corre-
`sponding to an actual IGF-1 concentration of 3.6 g/L was
`achieved, with an inclusion body size of 0.33 mm. The
`recombinant protein concentration was much higher
`than that obtained using constant-rate feeding. There
`was, however, no improvement in inclusion body size.
`This suggests the increase in total product is solely due
`to the increased biomass concentration. There are several
`possible reasons why we did not see an increase in the
`specific ratio of product to biomass. Further optimisation
`of the feed profile to restrict acetate below the inhibitory
`concentration may be necessary. Optimisation of other
`key parameters (e.g., IPTG concentration) may also be
`necessary, although previous tests suggest relative insen-
`sitivity (data not shown). We have previously observed
`translational
`limitation
`for this strong promoter
`(Jorgensen et al., 1997), so this is a likely limit to fur-
`ther productivity increases. To test this, a 3-stage fed-
`batch fermentation with yeast-extract-supplemented
`nutrient feed after induction was conducted.
`
`The fermentation profiles for exponential feeding with
`yeast-extract-supplemented nutrient are presented in
`Figure 3b. An OD600 of 163, corresponding to a dry
`cell weight of 56 g/L, was achieved. This was higher
`than that obtained using exponential feeding without
`yeast extract supplementation. Glucose accumulation
`was reduced to a level of 7.3 g/L. After induction,
`acetate gradually accumulated and reached a level of
`
`Biotechnology Letters · Vol 19 · No 11 · 1997
`
`1081
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`Figure 2
`
`The novel 3-stage feeding strategy.
`
`Figure 3 Fed-batch fermentation profiles using exponen-
`tial feeding. u, dry cell weight (DCW); r, acetate concen-
`tration; m, glucose concentration. (a) without yeast-extract
`supplementation; (b) with yeast-extract supplementation.
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`N.D. Wangsa-Wirawan et al.
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`4.4 mM at the end of fermentation. This is much lower
`than for the previous fermentations. A recombinant
`protein concentration of 3.7 g/L, corresponding to an
`IGF-1 concentration of 3.0 g/L, and an inclusion body
`size of 0.32 mm were achieved. Yeast extract supple-
`mentation did not improve inclusion body size. A lower
`recombinant protein level was also obtained. The pres-
`ence of yeast extract may enhance the growth of cells
`and thus reduce the rate of protein expression, or may
`simply alter the amino acid balance. This will influence
`the metabolic flux through amino acid pathways and
`may reduce overall protein expression.
`
`The novel 3-stage feeding procedure reported in this
`paper improved the productivity of IGF-1 fusion protein
`using recombinant E coli JM101. Glucose and, conse-
`quently, acetate accumulation were greatly decreased.
`Recombinant fusion protein concentration was increased
`from 2.6 g/L to 4.3 g/L, corresponding to an actual
`IGF-1 concentration of 3.5 g/L. However, inclusion body
`size was not altered by the novel feeding strategy nor by
`the supplementation of yeast extract. Further investiga-
`tion of factors altering inclusion body size and protein
`expression level, and further optimisation of the feeding
`strategy, has the potential to further increase the already-
`high IGF-1 concentration achieved in this study.
`
`Acknowledgment
`N.D. Wangsa-Wirawan thanks the CRC for Tissue
`Growth and Repair for partial support of this work
`through provision of a supplementary scholarship, and
`the University of Adelaide for support in the form of
`an OPRS award and a University of Adelaide
`Scholarship.
`
`References
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`Jorgensen, L, Thomas, CJ, O’Neill, BK and Middelberg, APJ
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`Received 28 July 1997
`Revisions requested 27 August 1997
`Revisions received 9 September 1997
`Accepted 9 September 1997
`
`1082
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