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Influence of Scale-Up on the Quality of

Recombinant Human Growth Hormone

F. Bylund,1 A. Castan,2 R. Mikkola,2 A. Veide,1 G. Larsson1
1
Centre for Bioprocess Technology, Department of Biotechnology, Royal
Institute of Technology (KTH), SE-100 44 Stockholm, Sweden; telephone:
+46-8-790-8738; fax: +46-8-790-9540; e-mail: gen.larsson@biotech.kth.se
2
Pharmacia & Upjohn AB, Process R & D, SE-112 87 Stockholm, Sweden
Received 25 July 1999; accepted 9 January 2000

Abstract: The aerobic fed-batch production of recombi- In this context, two significant industrial problems with
nant human growth hormone (rhGH) by Escherichia coli regard to the cultivation step of biological processes can be
was studied. The goal was to determine the production
and protein degradation pattern of this product during identified: (i) batch variations, especially on the large scale;
fed-batch cultivation and to what extent scale differences and (ii) lack of reproducibility when the scale is changed.
depend on the presence of a fed-batch glucose feed The latter refers to both the scale-up and scale-down situa-
zone. Results of laboratory bench-scale, scale-down tions and is introduced either when new processes are
(SDR), and industrial pilot-scale (3-m3) reactor produc-
tion were compared. In addition to the parameters of implemented or when old processes are being redesigned
product yield and quality, also cell yield, respiration, and/or optimized. The problems can be caused by a multi-
overflow, mixed acid fermentation, glucose concentra- tude of factors and presently there is no general scientific
tion, and cell lysis were studied and compared. The re- method to deal with them. However, some steps in creating
sults show that oxygen limitation following glucose
such a methodology have recently been taken.
overflow was the critical parameter and not the glucose
overflow itself. This was verified by the pattern of by- Although the reasons for batch variation have not been
product formation where formate was the dominating the topic of much interest in the scientific community there
factor and not acetic acid. A correlation between the ac- have been some important results found based on scaling
cumulation of formate, the degree of heterogeneity, and studies of biological processes during the last 15 years.
cell lysis was also visualized when recombinant protein
was expressed. The production pattern could be mim- Breakthroughs have been made in hydrodynamics and mix-
icked in the SDR reactor for all parameters, except for ing, modeling of two-phase gas/liquid biological processes
product quantity and quality, where 30% fewer rhGH- (Guillard, 1999), large-scale microenvironment measure-
degraded forms were present and where about 80% ments (Bylund et al., 1998, 1999; George, 1997; Larsson et
higher total yield was achieved, resulting in 10% greater
accumulation of properly formed rhGH monomer. © 2000
al., 1996; Manfredini et al., 1983; Oosterhuis and Kossen,
John Wiley & Sons, Inc. Biotechnol Bioeng 69: 119–128, 2000. 1984), and scale-down methodology studies (Oosterhuis et
Keywords: recombinant human growth hormone (rhGH); al., 1983, 1985). The latter area of study includes the use of
product quality; Escherichia coli fed-batch; substrate gra- scale-down reactors (SDRs) with various biological model
dients; scale-up; scale-down reactor (SDR) systems (Bylund et al., 1999; Fowler and Dunlop, 1989;
George et al., 1993, 1998; Larsson and Enfors, 1988;
INTRODUCTION Moes et al., 1985; Neubauer et al., 1995a,b; Sweere et al.,
1988a–c).
Production of proteins by recombinant technology has been In brief summary, these findings point to the existence of
the subject of much industrial interest due to the possibili- concentration gradients in reactors also on a quite moderate
ties of cutting production costs. However, production has reactor scale. That substrate gradients exist was to be ex-
been limited to a few overexpression systems of which pected, because substrates that are fed in limited amounts
knowledge with respect to process performance is limited. (glucose, oxygen) in fed-batch cultivation processes cannot
The complexity of the response of the catalyst to the sub- be immediately mixed to homogeneity in the reactor. Fur-
strate is also a significant factor. Furthermore, recombinant thermore, substrate concentration is approximately 10,000
protein processes suffer from the same general problems times higher near the feed inlet compared with the mean
during choice of production technique and scale-up as all reactor concentration and the dissipation pattern is a func-
other biologically and chemically based production sys- tion of the degree of mixing at the location of the feed point
tems. (Bylund et al., 1999; Larsson et al., 1996).
Presently, however, there are no hydrodynamic models or
Correspondence to: G. Larsson
translations into engineering parameters of the findings just
Contract grant sponsors: Swedish Centre for Bioprocess Technology referred to that can describe or directly influence the pat-
(CBioPT); Pharmacia & Upjohn AB terns of substrate gradients. An important observation in

© 2000 John Wiley & Sons, Inc.

shown to vary drastically between shake-flask and bioreac- sults can presently provide only a yes/no answer to the tor cultivation (Yang. the SDR set-up could only substrate concentration. an SDR was designed consisting of We chose to study the production of recombinant human two CSTRs connected in a loop. Even though the total amount of product tinguish the fed-batch effects. actor (Larsson et al. that would follow due to the increased respiration. and have also resulted in research to the point of describing changes in the gassing power (a in the kinetics of biological reactions toward transitions in quality parameter). all substrates are in excess accumulation. The large-scale reactor was growth hormone (rhGH) in Escherichia coli and the degra- thus approximated by one smaller reactor representing the dation products of this protein during aerobic fed-batch cul- stirring zone of comparatively high oxygen concentration and one comparatively larger reactor to mimic the bulk zone tivation on glucose in CSTRs of different scale. pense of the properly formed rhGH monomer.. This actor before dissipation (Larsson et al. uptake. geometry. VOL. that is. and organism. JULY 20. 1995).. the large-scale reactor. as outlined earlier. actor.. Both reactors were aerated. yield (g/g). tion is described. that is. However. However. metabolic byproduct formation. Formate. In trolled synthesis of a recombinant product protein under this context. high proteolytic protein turnover. out the cultivation. 1996). and increased with increased The second example involves the aerobic fed-batch cul. formation and protein quality (mainly in degradation pat- down-specific processes (to laboratory scale). 69. To the latter category inclusion-body formation. It can be assumed that uncon- question of whether scaling is likely to create a problem. and product proteolysis can be in- sent in the large reactors. and for oxygen the formic acid accumulation. in spite of use of the concept in the SDR set-up. Proteolysis is often a major problem and has been tant microenvironment features in a large reactor. The latter finding indicates 120 BIOTECHNOLOGY AND BIOENGINEERING. increased. small-scale laboratory reactor. re. 1983. Therefore. SDR re. Furthermore. 1999). These strate. 1998) and the aerobic results also showed that proteolytic degradation occurred in fed-batch CSTR (12 m3) cultivation of Escherichia coli pro. The goal From oxygen gradient patterns. tern) during cultivation. the effect of substrate variations on physiologi. and product forma. these are not based on tein production is productivity (g/g⭈h). This system is based on batch This would be seen in the pattern of metabolic byproduct technique characteristics. substrate considerable stress on the microorganism and can lead to. for glucose the acetic acid accumula- except for the oxygen that is fed to the process over time. huis et al. 2. The scale-up/scale-down research mimic some parameters of the fed-batch CSTR producing so far has led to that the biological studies of scaling was recombinant protein by cells of E. conditions in which the microenvironment varies can lead to cal parameters such as cell growth. The goal of this work was to detect variations in product Two distinct efforts have been made with regard to scale. for example. scale and was also detected much earlier the larger the scale. 2000 . This reactor consisted of degree of proteolysis was considerably lower in the SDR. tion. system has been successful in studies of baker’s yeast pro- Patterns of substrate oscillations have thus been identified duction with regard to bubble-column performance. 1996). was present already in the findings. namely oxygen limi- (CSTR) of 25 m3 with Gluconobacter suboxydans (Ooster.. if present. It was shown that gluconic acid The hypothesis of oxygen limitation as a main control production could be influenced by a variation of parameters factor in process performance. coli. the ing were used to design the SDR. In this context. To dis. respiration. and prod- any actual large-scale transitions. forming glucose gradients. “overemphasize” certain phenomena that are randomly pre- protein aggregation. tivation of Saccharomyces cerevisiae in a bubble-column This pattern could be mimicked by the SDR set-up. the degradation products accumulated at the ex- tration patterns and reactor hydrodynamic/kinetics model. However. However. could be a individual characteristic features of the actual process. This led the present range). cluded. The reactor of 215 m3 (George et al.this direction was that the frequency pattern of gradient the large reactor and the CSTR mimics the bulk zone of a oscillations was also detectable in the fluctuations of the comparatively low concentration (in the milligrams-per-liter turbulent velocity in the same location. even up as a key to scaling design. reason for the change in product quality during scaling. all scales and reactor configurations and increased through- ducing a recombinant protein (Bylund et al. 1985). one CSTR and one PFR (plug-flow reactor) in which the providing more of the correct rhGH form and less of the PFR mimics the feed zone (in the grams-per-liter range) of proteolytically degraded forms. was changes or production optimization were based on these verified. as result of the lack of a proper physical model for the impor. but rather are designed to uct quality. and modeling of was to modify existing SDRs according to this assumption. we wanted to deter- gies are based on actual measurements on a large scale and mine whether this pattern changed with scale and also if the use the same general concept. not acetic acid. tation. of lower concentration. is caused by a secondary batch cultivation in a continuous stirred tank reactor effect of high glucose concentration. done by making substrate transitions in various set-ups The major optimization parameters in recombinant pro- called scale-down reactors. but are designed from the fed-batch feed zone.. no large-scale parameter of control by glucose limitation in fed-batch operation. A further aim was to verify the hypothesis that the gra- The first investigation involves the scale-down of aerobic dient effect in CSTRs. NO. large-scale glucose concen. the feed zone is the sum of the investigators to conclude that intact “packages” of substrate volume of the glucose “packages” existing in the large re- of high concentration travel quite long distances in the re.

.the possibility of designing a new production technology for maintained due to sensitive equipment. stirrer speed up to 1000 rpm and 205 rpm in the lab-scale and pilot-scale bioreactors. Dissolved oxygen scaled up from control cultivations by a factor of 200. Its production is controlled by an inducible pro. four baffles. thus giving a zone of local earlier. A SDR consisting of two compartments with exchange Escherichia coli W3110. In one of the reactors. and the separately after sterilization. high glucose concentration. recombinant production processes. Breox (International Speciality volume of the PFR was 440 mL. sisting of 191 amino acids with two intramolecular disulfide 1999). lar to that measured for the lab-scale reactors during the ately started. The turbines. Pilot-scale cultivations were volumetrically trality by the addition of 25% NH3(aq). tension (DOT) in the bioreactor was monitored by a polaro. remaining space of a low bulk concentration (Bylund et al. to the feed inlet. (ii) a constant feed when the tor through a pipe in the top that had the orifice positioned estimated maximum oxygen transfer capacity of the biore. In these experiments tetracy. Ltd. the same amount of glucose was added per reactor graphic oxygen electrode and was controlled by varying the volume and hour in the different experiments. oxygen transfer rate (OTR) was 0.: SCALE-UP EFFECTS ON RECOMBINANT HUMAN GROWTH HORMONE PRODUCTION 121 . In the SDR experiments. and the production bioreactor laboratory scale. 1993). at the bottom.0 L). at the same level as the feed inlet but on the opposite side. this pipe was positioned was allowed to vary by ±5%. The inoculum reactor from the seed fermentor to the production bioreactor. The inoculum volume was about 10% of the final total volume. Sterilized mag. As a consequence. the stirrer speed was increased to 1200 rpm to avoid oxygen limitation in the CSTR. In pilot-scale experiments. As a reference. and a ring sparger at the bottom. The biomass concentration was determined as cell BYLUND ET AL. The flow char- moter. was used in bioreactor with enlarged glucose concentrations with limit- the cultivations performed. This coding for recombinant human growth hormone (rhGH) and reactor set-up is based on the view of a large-scale fed-batch tetracycline and ampicillin antibiotic resistance. On the had a total volume of 300 L. Medium components together with distilled water the PFR and showed a value of 0 at 3 to 4 h thereafter. the volume Chemicals.0 L.13 mol/L⭈h.1 L/min) was started 3 h after start of feed. two geometrically similar pro- Cultivation Procedure and Bioreactors duction lines were used. Growth was followed by biomass concentration measure- down experiments where increased pressure could not be ments. and one substrate reactor for Exponentially growing cells in batch mode were transferred ammonia and one for glucose feed. respectively. only low amounts of oxygen Cultivations were performed in a glucose mineral salt were present at high cell concentrations. and finally (iii) the feed rate was through a pipe and collected at the top. with a pBR322-derived plasmid flow was used for simulation of large-scale conditions. the glucose experiment was performed with the same construct as noted feed was added to the PFR inlet. the cell suspension was transferred via a 3 m3 (1. One exception was in the scale. DOT modifications of the components of the medium and trace was measured by a galvanic oxygen electrode at the top of elements.4 bar over pressure. the cell suspension diameter ratio of 1. This rhGH form is a 22-kDa protein con. glucose and low-oxygen concentration. The aeration rate Analytical Procedures was 1. a value simi- After the transfer procedure the glucose feed was immedi.0 L..7 and was equipped with two Rushton was pressed through a pipe connecting the bioreactors. UK).4 vvm (relative to the start volume) and the pressure at 0. Organism and Medium The volume increased during the feed phase up to 9. and antibiotics were added as in homogeneous lab-scale cultivations (7. All homogeneous control and inoculum lab-scale culti- MATERIALS AND METHODS vations were performed in a 15-L CF 3000 Chemap- Fermentor (Switzerland) CSTR with a start volume of 7. and the protein is secreted to the periplasm where the acteristics of the PFR have been described elsewhere disulfide bridges are formed. Temperature was controlled near the maximal value for and in the second reactor the sampling pipe was located next E. Thereby. In pilot-scale cultivations.4-m3 start volume). but where the gene coding for rhGH had been re. The bottom position decreased to 80% of its maximum value at the time of of this sampling pipe differed in location between pilot- induction. total volume in the two-compartment system was the same nesium sulfate. The feed profile consisted of three different conditions studied. The feed was constant thereafter. The two-compartment system consists of one CSTR bridges. each consisting of one inoculum and one production bioreactor. supplemented to this reactor. residence time 24 sec in the zone with combined high- tration of 590 g/L. (1997) with minor medium (1. coli growth and the pH was controlled at close to neu. ing amounts of oxygen in the feed zone or close to it and the cline was added. Glucose feed was added to the bioreac- phases: (i) an exponential feed. Because no additional air was moved. Media samples were taken from the bottom actor was approached. a pilot-scale (George et al. was added as needed fraction of medium in this reactor was 5% to 6% and the for foam control. The feed profile scale vessels. Southampton.. The were sterilized in the bioreactor at 121°C. The circulation of medium as described by Forsberg et al. The tank had a height-to- sterile flask. (Chemap Fermentor) in series with a PFR. The feed solution had a glucose concen. Consequently. trace elements.

batch phase and three fed-batch phases. cultivations as shown in Figure 2. yield of biomass. washing of the pellet with 5 mL of dis. was Four different variants of rhGH were separated by HIC about 0. acetate. The avoiding cell lysis. which uses magnetoacoustic and photoacoustic in. Brüel & (about 5 mg/L for E. at the time of induction. and drying overnight at 105°C manner. 0. a homogeneous control cultivation is in- rhGH were determined by hydrophobic interaction chroma- cluded in Figure 1. as done to inactivate substrate uptake by reducing the pH. was removed from the plasmid. with ultraviolet (UV) detection. 979 732). 2000 . The close posi- tion of sampling relative to feedpoint (see “Materials and RESULTS Methods”) is the most likely explanation for the fluctuations The cultivation parameters are generally shown in a set-up between samples and the occasionally higher concentrations of figures describing the three different experiments: (a) (up to 100 mg/L) measured in the pilot-scale bioreactor.4 g/g in all cultivations from RP-HPLC on a polymeric column (Ashipac ODP-50). and (clipped 142/ ing the production phase. Glucose samples were taken by rapid sampling excess glucose. The reactor volume and total amount of biomass were calculated at each sampling occasion as described by By. tained on a pilot scale (Fig. The corresponding biomass con- formate: Kit No. (b) heterogeneous SDR. 40 g/L. and (trisulfide Cys182–Cys189)-rhGH.4551 g. the results ob- frared spectroscopy. and ethylene-diamine tetra- comparison to further experiments with the SDR and pilot- acetic acid (EDTA) × 2H2O. (clipped 142/143)-rhGH tenance requirements at the end of cultivation and not due to (clip-2). formate. As a 0. dropped to 0. feed rate was reduced to its final value.9837 g. min at 4500 rpm. Concentrations of glucose. but expected. The oxygen and carbon dioxide content in the outlet gas The glucose concentration in a fed-batch process is ex- flow was simultaneously measured with a combined oxygen pected to be around the saturation constant for the organism and carbon dioxide analyzer (Monitor Type 1308. and DNA analysis lum biomass concentration or viability. des-Pro2)-rhGH. (1990).372 g. Biomass Growth teins. (des-Phe1)-rhGH (LMW). Duplicate samples were taken from the growth rate further declined after induction (25 h). 2. This was measured by centrifugation of homogeneous control. Samples for product analyses were taken from the pellet fraction of 5 mL of centrifuged cell suspension. 148 261. 2c) are more difficult to interpret because the glucose concentration varied. The extrac- tion method used releases the periplasmic fraction of pro. at pH the start of the process to approximately 20 h. This means that the reason for the generally (des-Phe1. 122 BIOTECHNOLOGY AND BIOENGINEERING. All cultivations were performed in the same principal tilled water. resulting in the corresponding exponential tubes containing an equal volume of perchloric acid (0. the decrease was more pronounced and tection at 230 nm: (des-Phe1)-rhGH (abbreviated LMW). Purity and quantity of scale reactors. that is. biomass. coli). peak B contained (Met(O)14)- decreased yield was due to the comparatively higher main- rhGH. and the biomass and formate were determined with Boehringer-Mannheim increased only slightly thereafter and eventually stopped kits (glucose: Kit No. 0. This was started and the specific growth rate declined successively. Extraction buffer consisted of (per liter): Tris-HCl. VOL.25 g/g during the same time period. the yield coefficient was further reduced dur- Cys189)-rhGH (abbreviated trisulfide).dry weight (CDW). the first constant feed phase M) as described by Larsson and Törnkvist (1996). After this. (trisulfide Cys182– experiments. The initial batch The supernatant from the first centrifugation was sterile. In with a decreasing salt gradient and detected with UV de- SDR experiments. the yield decreased and. as described by Gellerfors was made in the cultivation where the recombinant gene et al. In all rhGH (correctly formed rhGH). Tris-base. passing through four different phases. performed on a TSK phenyl 5PW column (Tosoh Haas) and The yield coefficient was 0. JULY 20. acetate: Kit No. After depletion of (cell lysis). Substrate Uptake and Respiration lund et al. This was also true for the present Kjær). This is supported by a multitude of tography (HIC) and by isocratic reverse-phase high- process data based on optical density measurements (results performance liquid chromatography (RP-HPLC). NO. HIC was not shown). After this 8. and deamidated rhGH. and (c) in- 3 × 5 mL of cell suspension in preweighed test tubes for 10 dustrial pilot-scale cultivations. some hours thereafter. 69.5. when the pilot-scale bioreactor. and peak C contained rhGH product formation.3 g/g in the pilot-scale and control cultivations.13 growth (␮ < ␮max). one before weighing. whereas the biomass increased linearly. and specific growth rate. phase varied between 2 and 5 h due to differences in inocu- filtered and used for acetate. point. The DNA content of media centration in the different experiments was of the order of samples was determined with Pico-Green. However. although a slightly higher Three peaks (A to C) were identified and characterized final yield of biomass was obtained compared with the other with RP-HPLC: peak A contained (Met(O)125)-rhGH and cultivations. Centrifugation was performed at 5000 rpm for 15 min Cell growth is represented in Figure 1 as total amount of at +4°C. (1998). 716 251. recentrifuging. the exponential glucose-limited feed phase of cell suspension from the bioreactor into preweighed test was started. The same principal observation 143)-rhGH (abbreviated clip-2).

not shown due to error in the analysis. (a) ment. it has been shown that formate is not Respiration is shown in Figure 2a–c as the yield coeffi. coli is grown under anaerobic acetate is formed in the beginning of the process. consumed as rapidly as acetate when returning to a region cients of oxygen and carbon dioxide per added glucose with a high level of oxygen (Cleland. later re- or oxygen-limiting conditions (mixed acid fermentation) as placed by formate formation. Glucose concentration (milligrams per liter) and respiration data shown as yield coefficient of oxygen and carbon dioxide per glucose Figure 1. 1996). the other hand. increasing oxygen-limiting conditions. and (3) 〫. Furthermore. Specific growth rate marked as (1) ⽧. (2). Of these me- (YO2/S and YCO2/S. Generally. The general trend was a relatively tabolites. Glycolytic Byproducts Acetate and formate were produced in all cultivations. as yield coefficient of oxygen marked as: (1) 䊉. (b) scale-down experiments. (2) 䊊.: SCALE-UP EFFECTS ON RECOMBINANT HUMAN GROWTH HORMONE PRODUCTION 123 . (2) tively. Yield coefficient marked as (1) 䉱. Respiration data for pilot-scale experiments (1) and (3) are experiment in which the recombinant gene was removed from the plasmid. However. and (c) pilot-scale experiments. This is a further verification of gradient formation in bio. and (3) +. Biomass marked and (3) and marked with different symbols. detected before formate in a batch phase when exposed to tically after induction. a value of 0. Pilot-scale cultivation (3) corresponds to the 䉭. but at different rates and at different times (Fig. Acetate level was dependent on process BYLUND ET AL. Glucose concentration as well as (1) 䊉. respectively). respectively. well as when growing aerobically in the presence of excess Acetate was accumulated during the batch and the expo- glucose (overflow metabolism) (Lee. on nential feed phases. Acetate is produced when E. and (3) 䉱. g/g). (a) Bench-scale control experi- mass per glucose added (YX/S. Formate. 1988). and (3) 䊏. cate samples are shown for the glucose concentration in pilot-scale experi- scale experiments. respec- (2) 䉭. (2). 3). acetate was shown by Vollbrecht (1982) to be constant value during the growth phase that increased dras. Figure 2. and (3) X. Yield coefficient of carbon dioxide marked as: (1) 䊊. Repeated cultivations in the different reactors are numbered (1). which was never measured during the cultiva- tions in the present investigation. This indicates higher respiration dur. Repeated cultivations in the different reactors are num. and (3) and marked with different symbols. bered (1). Dupli- Bench-scale control experiment. (b) scale-down experiments. yield coefficient of bio. Cell growth shown as biomass (au). and (c) pilot. respectively. added (YO2/S and YCO2/S g/g. the con- ing the production of the target protein and when growth centration of the latter increased first after the DOT showed declines. (2) 䊏. and specific growth rate (␮/␮max). respectively. g/g). is only produced during mixed acid fermen- reactors at the 3-m3 scale. tation. (2) 䊐. respectively. ments.

(2) 䊏. and (3) and marked with different symbols. the longer the period in the phase. JULY 20. Acetate was mainly consumed during the first hours of the subsequent constant feeding phase. respectively. Formate con- centration marked as (1) 䊉. the overall result of the SDR cultivations gave the same byproduct pattern as for the other cultivations (Fig. respectively. process. the differences in biomass between cultiva- tions cannot account for the differences in formate accumu- lation. Figure 4. is shown in Figure 6a. The higher cell lysis in scale-down experiments may partially explain the lower biomass in these cultivations. In pilot-scale cultivations. the results were inter- mediate but accumulation was more rapid after induction up to the levels obtained at the laboratory scale. which shows that production of rhGH is an additional source for cell lysis. respectively. including correct GH monomer and its variants. and (3) 䉭. NO. and (c) pilot- scale experiments. because acetate accumulated scale cultivations (1) 䊉. and was lowest in the control experiment. Pilot-scale culti- only in the beginning of the cultivation. Formate and acetate accumulation during cultivations. in relation to induction is shown in Figure 5. It has been shown in the literature that overflow metabolism occurs at substrate uptake rates comparable to a growth rate of 0. (a) Bench-scale control experiment. Cell Lysis The amount of DNA in the medium. and (3) 䉱. (2). This byproduct accumulated rapidly up to a concen- tration of 500 mg/L. time in the batch phase. Product Formation The total yield of product. Cultivations marked as: control 䊊. Acetate con- centration marked as (1) 䊊. SDR 䊐. VOL. and to avoid acetate formation the flux should be kept below this value (Lee. As can be seen from Figure 4. Comparison of the different reactors with respect to (a) cell growth shown as biomass (au). and repeated pilot- cells compared with formate. and (b) formate concentration (milligrams indicating that acetate was more rapidly taken up by the per liter). (2) 䊐. which is comparable to cell lysis. 2. 2000 . However. and (3) 䉱.35 h−1 (in minimal medium). The general trend is Figure 3. 4). The ac- cumulation rate increased after induction in all cultivations. (2) 䊏. where the higher the concentration measured. Formate was produced after induction on a laboratory scale. Pilot-scale cultivation (3) corresponds to the experiment in which the recombinant gene was removed from the plasmid. 124 BIOTECHNOLOGY AND BIOENGINEERING. In the SDR set-up. 1996). Formate increased vation (3) corresponds to the experiment in which the recombinant gene up to a value of 1000 mg/L in the latter part of the SDR was removed from the plasmid. Repeated cultivations in the different reactors are num- bered (1). 69. The reason why acetate was also produced during the late exponential feed phase is probably due to the high specific growth rate. the pilot-scale experiment in which the recombi- nant gene had been removed was similar to the lab-scale control experiment. followed by pilot-scale experi- ments. Notably.30 to 0. both metabolites were produced in the PFR at all times (results not shown). (b) scale-down experiments. These results show that the amount of DNA was highest in scale-down cultivations.

respectively. where these experiments showed comparable results. SDR 䊐 and 䉭. The amount of DNA is correlated with the degree of cell lysis in the experiments. Processes for recombinant protein production are often The comparison of pilot-scale and scale-down two.8. The dominating form here was clip-2 and the differences in the amount of trisulfide. 75. inoculum were found to have a pronounced effect on a BYLUND ET AL. SDR. dissolved oxygen showed a value of 30% air in the stirred lot-scale productions. the amounts of formate were detected even though the proteolysis between the laboratory-scale control and the pi. However. The amounts of proteolytically degraded forms of the Figure 6.8 ± 11.” which includes LMW. scale-down. and 41.Figure 5. variations in biomass concentration of the found in the SDR. The inoculum prepa- ration is comparatively simpler in the laboratory. clip-2. This was concluded constant growth as well as declining growth). clip-2. compartment experiments shows that the ruling regime on a such as growth phase (inoculum procedure. for instance.1. and further points to the difficulties of the scale-up procedure. The final product mean values per cell dry weight in lab-scale (con- trol and SDR) and pilot-scale experiments were 64. and 72. but was exaggerated. difficult to control due to the number of different phases. was lowest in marked: control. tank reactor.2 ± 1. Figure 6b shows rect GH monomer and its variants. (a) Total amount of rhGH per biomass (au) that includes cor- product were different in the experiments. (1) scale-down experiments. respectively.6 ± 9. Cultivations marked as: control 䊊. Values are shown with 95% confidence interval. (Met(O)14)-rhGH. There were no apparent differences in tably.0 ± 1. In the present inves- cumulation could be simulated.4. DNA concentration in the medium (micrograms per milliliter). with about 30% less “B” in the 䊏 and (2) 䊐. a linear increase of product but at a lower level and rate in the pilot-scale cultivations.: SCALE-UP EFFECTS ON RECOMBINANT HUMAN GROWTH HORMONE PRODUCTION 125 . Cultivations are (Met(O)14)-rhGH. at the end of the cultiva- tions. that the amount of “B. and deamidated rhGH. there was a dif.9. tigation.8% in the control. and deamidated rhGH. respectively (val- ues expressed with 95% confidence interval). phase (time of induction). and re- peated pilot-scale cultivations (1) 䊉. 70. (2) 䊏. respectively. (2) 䉲 and (3) ⽧. production from the findings that the pattern of formate and DNA ac. and (c) amount of correct GH monomer (% of total GH). 76. Pilot- scale cultivation (3) corresponds to the experiment in which the recombi- nant gene was removed from the plasmid. (1) 䉱. followed by the pilot-scale and the lab. 6c) was. the cultivations passed through different control ference in the absolute value. Initially. and pilot scale. Comparisons of protein production show that the substrate oscillations decreased the accumulation of pro- DISCUSSION teolytically degraded forms of rhGH. This may be partially attribut- able to the quality of the inoculum. and harvest.2 au.0 ± 1. No- (result not shown). (1) 䊉. and pilot-scale experiments. were scale control experiments.7 ± 8. final sample. and (3) 䉱. exponential and pilot scale is mimicked in the SDR. where the highest values were regimes. The amount of correct monomer (Fig. (b) amount of “B” (% of total GH). which includes LMW. The pilot-scale re- small—only about 1% higher during pilot-scale cultivations actor was mimicked in the SDR.

In these cultivations. the growth declined in this cultivation The amount of DNA in the medium. 1996). removing the recombinant gene glucose-limiting conditions. The results point to the dynamics of recombinant saturation. 69. the pilot-scale cultivation was similar to con- ferent pattern with regard to glucose concentration. The rate of protein synthesis also grown on glucose (0. mate concentration increased first after induction. during the production phase. were most likely not the rea- from the plasmid gave a similar low-yield coefficient of 0. This indicates that the cell physiology differed in eters may interfere: DNA concentration reaches a stable these cultivations as a result of the longer batch phase.. Furthermore. in this specific etate formation. gree of oxygen limitation was influenced by both the induc- similar biomass growth was generally obtained. Strandberg et al. be avoided by choosing a specific growth rate was >30% air saturation. production of the recombinant protein had. acetate strongly influenced by the feed-controlled regime. the due to cell lysis. mass yield (YX/S) was 0. the plasmid. cient. Before induction. At the point of induction. the cultiva. 1998). These results clearly show reduction took place mainly during the production phase. This byproduct formation occurred de- oxygen demand and carbon dioxide production per glucose spite the fact that dissolved oxygen tension was >30% air added. etate concentrations was obtained during glucose starvation uct. the creased well before induction. even though the enzymes for the corresponding lower in all the cultivations than the normal value for E. This indicates that the de- tion (YO2/S and YCO2/S). The reason reactor (Fig. However. Therefore. 1). this did not lead to increased degradation of prod. The formation of acetate during the exponential feed phase ing several effects: (i) oxygen limitation. 1999. lower than 0. shift from aerobic conditions to anaerobiosis is in part simi- lowing turn on/off of metabolism. and (ii) lower product formation tion pattern differed in control and one pilot-scale cultiva- than expected. the increase in cell lysis followed by consumption during the constant feed (Fig. several param. no increase in formate or ac- However. feed rate. After induction. Therefore. including biomass growth and ac. concentration during the batch and exponential feed phase served. 2). 1c). showed a strong correlation with the ac- 126 BIOTECHNOLOGY AND BIOENGINEERING. which probably result in severe growth rate.number of parameters. but pected pattern for most experiments. 1998. seen in all cultivations is the appearance of local low oxy- nance effects (reduced biomass). increased pro. The bio.. JULY 20. 2. 1998. that is. although different depending on scale. batch phase was longer in these experiments. and in this study was in biomass yield was larger in the scale-down experiments. NO. however. This was seen in pilot-scale cultivations as case.. The acetate and limitation of oxygen because of the increased demand. when the recombinant gene was removed from on a pilot scale and in a control experiment showed a dif. (Nyström. It has been shown by conditions and was on the order of minutes to hours. because was not consumed at the same level during the early con- any overfeed of glucose might result in a rapid accumula. the low growth rate and the reduced plasmid copies results in a decreased cell yield and specific feed rate in our experiments. however.4 g/g in the cultivations during the The formate concentration in scale-down experiments main part of the growth phase. g/g. but this value was succes. was severely interfering with the process performance. acetate. tion was shifted to feed-controlled growth where the effect The acetate concentration in the medium showed an ex- of overflow metabolism (acetate) was most dominant. and a 100% increase in gen concentrations.3 to 0. George et al. although the DOT can. The explanation why the biomass yield coefficient was conditions. The presence of substrate been shown during IPTG (isopropyl-␤-D-thiogalactoside) gradients and oscillating conditions has been shown in other and temperature induction of other recombinant proteins large-scale bioreactors by several investigators (Bylund et (Bylund et al. There was. was consumed immediately after induction and decrease in Furthermore. value. it has been shown in batch for this was probably the stress introduced due to the fluc. 3b and 5). On the Despite the fact that recombinant cultivations performed other hand. The decrease al. experiments that the synthesis of specific proteins during a tuations of glucose and oxygen concentrations and the fol. coli pathways were present. The most likely reason for the accumulation of formate teolytical breakdown of the product. Furthermore. A similar maintenance effect has for substrates fed to the process. After this point the cultivations were tion compared with the others. a difference in that the tion of the overflow metabolite acetate to lethal amounts. showed the highest value and the concentration also in- sively decreased some hours before induction. re- Seo and Bailey (1985) that carrying an increased number of spectively. but at a lower rate (Fig. giv. 1994).35 h−1 (Lee. tion as well as its scale dependence. indicated by the variance of the glucose values in the pilot resulting in a 6% to 10% lower final biomass. VOL. increasing mainte. The increased level of formate was obtained ear- processes and the need for efficient feedback control where lier in pilot-scale cultivations when compared with homo- critical parameters need to be known and measured in a geneous lab-scale experiments (Fig.. an increased oxygen limitation due to scale effects was also clearly ob.5 g/g) is probably the high plasmid differed for a shift to anaerobiosis and glucose starvation burden under uninduced conditions. 4b) in which the for- real-time scale. 1994). that the accumulation of formate in large-scale bioreactors This decrease occurred due to increased maintenance when can be predicted by introducing an oscillating environment growth declined (Fig. 3). which is released as in the others. which was seen from the increas. therefore. trol experiments in this respect. lar to the response of cells shifted to glucose starvation tion of formate and also higher cell lysis (Figs. and formate accumulation. respira. which caused the forma.4 sons for the production of formate. stant feed. 2000 . The acetate forma- ing amounts of formate. a minor contribution to the decrease in yield coeffi- well as on a laboratory scale.

the results show that the concentration of oxygen degradation of high-energy internal products. growth hormone is not cleaved by an longer and/or relaxation times shorter in the SDR. However. especially for the periplasm. It is also critical to follow and control the DNA change with cultivation time due to shear damage. The concentration of some recombinant performed all of the small-scale experiments. At first sight.oxy- cose feed. proteolysis of a specific target protein when accumulated in But.e. on residence time distribution. are more productive. etate did not accumulate. gen concentration in the two-compartment reactor. the latter lactate. fol. This degradation is assumed to be due to glucose starvation. However. However. most likely.. It is known that ATP-dependent proteolysis formate concentration and cell lysis. a higher DOT is needed to avoid mixed acid fermentation concentration reduces the amounts of otherwise formed pro- products such as formate. 1995). succinate. This means that oxygen limitation periods are probably However. This was not found in absolute values. there was a significant difference in Better control will result in the same cell physiology at the product accumulation and product quality of the rhGH be- start of the production phase. the cells vvm volume air per reactor volume and minute (L/L⭈min) BYLUND ET AL. On the one hand. sub- Tyr(143). because the cell mass is decreased and the cell lysis mand might have caused the formate formation and cell is increased by the oscillations. of increasing numbers of staphylococcal protein A (SPA) is reduced when glucose from laboratory control. 3 and 5). A future energy-dependent protease because the product is accumu. This cleavage site ever. in Figure 5.: SCALE-UP EFFECTS ON RECOMBINANT HUMAN GROWTH HORMONE PRODUCTION 127 . Therefore. ethanol. concentration is kept low in the medium (Yang.” with emphasis on clip-2). there was a trend in the experiments. It may therefore be accumulation interdependency. production of acetate/formate and the proteolytic enzymes (Gellerfors. that is that the oxygen limitation following the high glucose is. the neous laboratory-scale and pilot-scale cultivations. the conclusion is that. especially after induction. The reason could be that lysis. that the amount of cell cultivations was in the accumulation of formate and not in lysis follows the degree of exposure to heterogeneity in acetate. occur in the cytoplasm before or during the translocation to the periplasm and the formation of intact The authors thank Anders Persson (Pharmacia & Upjohn) who disulfide bridges. even though ac- recombinant protein-producing cultivations. points to GH monomer increased by approximately 10% when the the need for efficient real-time feedback control of the glu- cells were exposed to an oscillating high-glucose. coli is. The complex dynamics of tween experiments. On the other hand. the substrate feed with the same accuracy in all scales. and teases that degrade the product. The in- The oscillations in the scale-down reactor result in better creased quantity of GH monomer was comparable to a production and product quality compared with homoge- lower occurrence of proteolytically cleaved forms (i. low-oxygen concentrations. it might have been formed but also erogeneous environment is thought to be in the SDR. 1990).e. be based on an SDR with a broader lated in the periplasm. the cells have a longer relaxation time when going from high to presence of DNA due to induction could decrease the dif. How- amount of “B.. consumed again when not exposed to zones with high- lowed by the pilot-scale reactor and the homogeneous lab. process results and thereby batch variations should most fers. This means that fusion rate of oxygen to the cell surface. The most het.. This is since the final formate concentration is similar in control and pilote. likely be avoided. Carbon-starved E. acetate. which in turn released DNA. it should be noted that. low glucose concentration than vice versa. glucose. In longer time and hence no signals of severe limitation call for any case. therefore. and cell lysis (i. the other hand. in spite of the CSTR continuous stirred tank reactor increased heterogeneity in the microenvironment. It was found that the amount of correct recombinant processes. because the greatest difference between One may observe. low. it seems to be the higher rate of release of DNA that causes the earlier accumulation CONCLUSIONS of formate rather than its absolute value. to SDR production. The amount of glucose added per reactor volume and time must be adapted to the actual amount of tional to the formate accumulation might be that the size of biomass at feed start. DOT dissolved oxygen tension (%) OTR oxygen transfer rate (mol/L⭈h) known to increase the degradation of the bulk protein at an PFR plug-flow reactor average rate from about 1% to 2% to 4% to 5% (Reeve et SDR scale-down reactor al. seems more likely. proteins in batch cultivations has been shown to reach a maximum value just before the glucose is exhausted and NOMENCLATURE declines thereafter. design might. Precultivation stages must be reproducible to give reliable scale experiments while the final DNA concentration dif. thus causing an the cells “imagine” being in a richer environment for a increased apparent saturation constant for this substrate. A reason why the DNA concentration is not propor. An alternative in the reactor can be rate limiting even at higher values. which is an exposed domain easily accessible to strate uptake rate. growth. scale reactor (control).cumulation of formate (Figs. During the process. a quali- a connection between the degree of substrate gradients and tative resemblance). The proteolytic cleavage could. the SDR mimics patterns of substrate and metabolite has been identified to amino acid residues Thr(142)– accumulation of the pilot-scale process for growth rate. to pilot-scale. for instance. this is at the expense of oxygen limitation caused by the increased respiratory de. 1984).

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