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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: 119128, 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.,
1988ac).
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.


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. This led the present range). In this context, 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- actor (Larsson et al., 1996). Both reactors were aerated. This
actor before dissipation (Larsson et al., 1996). system has been successful in studies of bakers yeast pro-
Patterns of substrate oscillations have thus been identified duction with regard to bubble-column performance, even up
as a key to scaling design, 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). However, the SDR set-up could only
substrate concentration. 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. coli.
done by making substrate transitions in various set-ups
The major optimization parameters in recombinant pro-
called scale-down reactors. However, these are not based on
tein production is productivity (g/gh), yield (g/g), and prod-
any actual large-scale transitions, but rather are designed to
uct quality. To the latter category inclusion-body formation,
overemphasize certain phenomena that are randomly pre-
protein aggregation, and product proteolysis can be in-
sent in the large reactors, as outlined earlier. Therefore, as
result of the lack of a proper physical model for the impor- cluded. Proteolysis is often a major problem and has been
tant microenvironment features in a large reactor, SDR re- shown to vary drastically between shake-flask and bioreac-
sults can presently provide only a yes/no answer to the tor cultivation (Yang, 1995). It can be assumed that uncon-
question of whether scaling is likely to create a problem. In trolled synthesis of a recombinant product protein under
this context, the effect of substrate variations on physiologi- conditions in which the microenvironment varies can lead to
cal parameters such as cell growth, respiration, substrate considerable stress on the microorganism and can lead to,
uptake, metabolic byproduct formation, and product forma- for example, high proteolytic protein turnover.
tion is described. The goal of this work was to detect variations in product
Two distinct efforts have been made with regard to scale- formation and protein quality (mainly in degradation pat-
down-specific processes (to laboratory scale). These strate- tern) during cultivation. Furthermore, 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, but are designed from the fed-batch feed zone, forming glucose gradients, could be a
individual characteristic features of the actual process, re- reason for the change in product quality during scaling.
actor, and organism. A further aim was to verify the hypothesis that the gra-
The first investigation involves the scale-down of aerobic dient effect in CSTRs, if present, is caused by a secondary
batch cultivation in a continuous stirred tank reactor effect of high glucose concentration, namely oxygen limi-
(CSTR) of 25 m3 with Gluconobacter suboxydans (Ooster- tation, that would follow due to the increased respiration.
huis et al., 1983, 1985). This system is based on batch This would be seen in the pattern of metabolic byproduct
technique characteristics; that is, all substrates are in excess accumulation; that is, for glucose the acetic acid accumula-
except for the oxygen that is fed to the process over time. tion, and for oxygen the formic acid accumulation. The goal
From oxygen gradient patterns, geometry, and modeling of
was to modify existing SDRs according to this assumption.
the large-scale reactor, an SDR was designed consisting of
We chose to study the production of recombinant human
two CSTRs connected in a loop. 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.
of lower concentration. 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, in spite of use of the concept
in the SDR set-up. However, no large-scale parameter of control by glucose limitation in fed-batch operation, was
changes or production optimization were based on these verified. Formate, not acetic acid, was present already in the
findings. small-scale laboratory reactor, and increased with increased
The second example involves the aerobic fed-batch cul- scale and was also detected much earlier the larger the scale.
tivation of Saccharomyces cerevisiae in a bubble-column This pattern could be mimicked by the SDR set-up. The
reactor of 215 m3 (George et al., 1998) and the aerobic results also showed that proteolytic degradation occurred in
fed-batch CSTR (12 m3) cultivation of Escherichia coli pro- all scales and reactor configurations and increased through-
ducing a recombinant protein (Bylund et al., 1999). To dis- out the cultivation. Even though the total amount of product
tinguish the fed-batch effects, large-scale glucose concen- increased, the degradation products accumulated at the ex-
tration patterns and reactor hydrodynamic/kinetics model- pense of the properly formed rhGH monomer. However, the
ing were used to design the SDR. This reactor consisted of degree of proteolysis was considerably lower in the SDR,
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. The latter finding indicates

120 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 69, NO. 2, JULY 20, 2000
the possibility of designing a new production technology for maintained due to sensitive equipment. As a consequence,
recombinant production processes. the stirrer speed was increased to 1200 rpm to avoid oxygen
limitation in the CSTR.
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 7.0 L.
Organism and Medium The volume increased during the feed phase up to 9.0 L.
A SDR consisting of two compartments with exchange
Escherichia coli W3110, with a pBR322-derived plasmid flow was used for simulation of large-scale conditions. 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, was used in bioreactor with enlarged glucose concentrations with limit-
the cultivations performed. In these experiments tetracy- ing amounts of oxygen in the feed zone or close to it and the
cline was added. This rhGH form is a 22-kDa protein con- remaining space of a low bulk concentration (Bylund et al.,
sisting of 191 amino acids with two intramolecular disulfide 1999). The two-compartment system consists of one CSTR
bridges. Its production is controlled by an inducible pro- (Chemap Fermentor) in series with a PFR. The flow char-
moter, and the protein is secreted to the periplasm where the acteristics of the PFR have been described elsewhere
disulfide bridges are formed. As a reference, a pilot-scale (George et al., 1993). In the SDR experiments, the glucose
experiment was performed with the same construct as noted feed was added to the PFR inlet, thus giving a zone of local
earlier, but where the gene coding for rhGH had been re- high glucose concentration. Because no additional air was
moved. supplemented to this reactor, only low amounts of oxygen
Cultivations were performed in a glucose mineral salt were present at high cell concentrations. The circulation of
medium as described by Forsberg et al. (1997) with minor medium (1.1 L/min) was started 3 h after start of feed. DOT
modifications of the components of the medium and trace was measured by a galvanic oxygen electrode at the top of
elements. Medium components together with distilled water the PFR and showed a value of 0 at 3 to 4 h thereafter. The
were sterilized in the bioreactor at 121C. Sterilized mag- total volume in the two-compartment system was the same
nesium sulfate, trace elements, and antibiotics were added as in homogeneous lab-scale cultivations (7.0 L), and the
separately after sterilization. Breox (International Speciality volume of the PFR was 440 mL. Consequently, the volume
Chemicals, Ltd., Southampton, UK), was added as needed fraction of medium in this reactor was 5% to 6% and the
for foam control. The feed solution had a glucose concen- residence time 24 sec in the zone with combined high-
tration of 590 g/L. glucose and low-oxygen concentration.
In pilot-scale experiments, two geometrically similar pro-
Cultivation Procedure and Bioreactors duction lines were used, each consisting of one inoculum
and one production bioreactor, and one substrate reactor for
Exponentially growing cells in batch mode were transferred ammonia and one for glucose feed. The inoculum reactor
from the seed fermentor to the production bioreactor. On the had a total volume of 300 L, and the production bioreactor
laboratory scale, the cell suspension was transferred via a 3 m3 (1.4-m3 start volume). The tank had a height-to-
sterile flask. In pilot-scale cultivations, the cell suspension diameter ratio of 1.7 and was equipped with two Rushton
was pressed through a pipe connecting the bioreactors. The turbines, four baffles, and a ring sparger at the bottom. The
inoculum volume was about 10% of the final total volume. oxygen transfer rate (OTR) was 0.13 mol/Lh, a value simi-
After the transfer procedure the glucose feed was immedi- lar to that measured for the lab-scale reactors during the
ately started. The feed profile consisted of three different conditions studied. Glucose feed was added to the bioreac-
phases: (i) an exponential feed; (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- at the bottom. Media samples were taken from the bottom
actor was approached; and finally (iii) the feed rate was through a pipe and collected at the top. The bottom position
decreased to 80% of its maximum value at the time of of this sampling pipe differed in location between pilot-
induction. The feed was constant thereafter. The feed profile scale vessels. In one of the reactors, this pipe was positioned
was allowed to vary by 5%. at the same level as the feed inlet but on the opposite side,
Temperature was controlled near the maximal value for and in the second reactor the sampling pipe was located next
E. coli growth and the pH was controlled at close to neu- to the feed inlet. Pilot-scale cultivations were volumetrically
trality by the addition of 25% NH3(aq). Dissolved oxygen scaled up from control cultivations by a factor of 200.
tension (DOT) in the bioreactor was monitored by a polaro- Thereby, 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.
stirrer speed up to 1000 rpm and 205 rpm in the lab-scale
and pilot-scale bioreactors, respectively. The aeration rate
Analytical Procedures
was 1.4 vvm (relative to the start volume) and the pressure
at 0.4 bar over pressure. One exception was in the scale- Growth was followed by biomass concentration measure-
down experiments where increased pressure could not be ments. The biomass concentration was determined as cell

BYLUND ET AL.: SCALE-UP EFFECTS ON RECOMBINANT HUMAN GROWTH HORMONE PRODUCTION 121
dry weight (CDW). This was measured by centrifugation of homogeneous control; (b) heterogeneous SDR; and (c) in-
3 5 mL of cell suspension in preweighed test tubes for 10 dustrial pilot-scale cultivations.
min at 4500 rpm, washing of the pellet with 5 mL of dis- All cultivations were performed in the same principal
tilled water, recentrifuging, and drying overnight at 105C manner, passing through four different phases; that is, one
before weighing. batch phase and three fed-batch phases. The initial batch
The supernatant from the first centrifugation was sterile- phase varied between 2 and 5 h due to differences in inocu-
filtered and used for acetate, formate, and DNA analysis lum biomass concentration or viability. After depletion of
(cell lysis). Glucose samples were taken by rapid sampling excess glucose, the exponential glucose-limited feed phase
of cell suspension from the bioreactor into preweighed test was started, resulting in the corresponding exponential
tubes containing an equal volume of perchloric acid (0.13 growth ( < max). After this, the first constant feed phase
M) as described by Larsson and Tornkvist (1996). This was started and the specific growth rate declined successively, as
done to inactivate substrate uptake by reducing the pH, but expected, whereas the biomass increased linearly. The
avoiding cell lysis. Duplicate samples were taken from the growth rate further declined after induction (25 h), when the
pilot-scale bioreactor. Concentrations of glucose, acetate, feed rate was reduced to its final value, and the biomass
and formate were determined with Boehringer-Mannheim increased only slightly thereafter and eventually stopped
kits (glucose: Kit No. 716 251; acetate: Kit No. 148 261; some hours thereafter. The corresponding biomass con-
formate: Kit No. 979 732). The DNA content of media centration in the different experiments was of the order of
samples was determined with Pico-Green. 40 g/L.
Samples for product analyses were taken from the pellet
fraction of 5 mL of centrifuged cell suspension. The extrac-
tion method used releases the periplasmic fraction of pro- Biomass Growth
teins. Centrifugation was performed at 5000 rpm for 15 min
Cell growth is represented in Figure 1 as total amount of
at +4C. Extraction buffer consisted of (per liter): Tris-HCl,
biomass, yield of biomass, and specific growth rate. As a
0.9837 g; Tris-base, 0.4551 g; and ethylene-diamine tetra-
comparison to further experiments with the SDR and pilot-
acetic acid (EDTA) 2H2O, 0.372 g. Purity and quantity of
scale reactors, a homogeneous control cultivation is in-
rhGH were determined by hydrophobic interaction chroma-
cluded in Figure 1. 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). HIC was
not shown).
performed on a TSK phenyl 5PW column (Tosoh Haas) and
The yield coefficient was 0.4 g/g in all cultivations from
RP-HPLC on a polymeric column (Ashipac ODP-50), at pH
the start of the process to approximately 20 h. After this
8.5, with ultraviolet (UV) detection.
point, the yield decreased and, at the time of induction, was
Four different variants of rhGH were separated by HIC
about 0.3 g/g in the pilot-scale and control cultivations. In
with a decreasing salt gradient and detected with UV de-
SDR experiments, the decrease was more pronounced and
tection at 230 nm: (des-Phe1)-rhGH (abbreviated LMW);
dropped to 0.25 g/g during the same time period. In all
rhGH (correctly formed rhGH); (trisulfide Cys182
experiments, the yield coefficient was further reduced dur-
Cys189)-rhGH (abbreviated trisulfide); and (clipped 142/
ing the production phase. The same principal observation
143)-rhGH (abbreviated clip-2), as described by Gellerfors
was made in the cultivation where the recombinant gene
et al. (1990).
was removed from the plasmid, 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. This means that the reason for the generally
(des-Phe1, des-Pro2)-rhGH; peak B contained (Met(O)14)-
decreased yield was due to the comparatively higher main-
rhGH, (des-Phe1)-rhGH (LMW), (clipped 142/143)-rhGH
tenance requirements at the end of cultivation and not due to
(clip-2), and deamidated rhGH; and peak C contained rhGH
product formation.
and (trisulfide Cys182Cys189)-rhGH.
The reactor volume and total amount of biomass were
calculated at each sampling occasion as described by By- Substrate Uptake and Respiration
lund et al. (1998).
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, Bruel & (about 5 mg/L for E. coli). This was also true for the present
Kjr), which uses magnetoacoustic and photoacoustic in- cultivations as shown in Figure 2. However, the results ob-
frared spectroscopy. tained on a pilot scale (Fig. 2c) are more difficult to interpret
because the glucose concentration varied. 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.

122 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 69, NO. 2, JULY 20, 2000
Figure 2. Glucose concentration (milligrams per liter) and respiration
data shown as yield coefficient of oxygen and carbon dioxide per glucose
Figure 1. Cell growth shown as biomass (au), yield coefficient of bio- added (YO2/S and YCO2/S g/g, respectively). (a) Bench-scale control experi-
mass per glucose added (YX/S, g/g), and specific growth rate (/max). (a) ment, (b) scale-down experiments, and (c) pilot-scale experiments. Dupli-
Bench-scale control experiment, (b) scale-down experiments, and (c) pilot- cate samples are shown for the glucose concentration in pilot-scale experi-
scale experiments. Repeated cultivations in the different reactors are num- ments. Repeated cultivations in the different reactors are numbered (1), (2),
bered (1), (2), and (3) and marked with different symbols. Biomass marked and (3) and marked with different symbols. Glucose concentration as well
as (1) , (2) , and (3) , respectively. Yield coefficient marked as (1) , as yield coefficient of oxygen marked as: (1) , (2) , and (3) , respec-
(2) , and (3) +, respectively. Specific growth rate marked as (1) , (2) tively. Yield coefficient of carbon dioxide marked as: (1) , (2) , and (3)
, and (3) X, respectively. Pilot-scale cultivation (3) corresponds to the , respectively. Respiration data for pilot-scale experiments (1) and (3) are
experiment in which the recombinant gene was removed from the plasmid. not shown due to error in the analysis.

This is a further verification of gradient formation in bio- the other hand, is only produced during mixed acid fermen-
reactors at the 3-m3 scale. tation. Furthermore, it has been shown that formate is not
Respiration is shown in Figure 2ac as the yield coeffi- 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, 1988). Of these me-
(YO2/S and YCO2/S, g/g). The general trend was a relatively tabolites, acetate was shown by Vollbrecht (1982) to be
constant value during the growth phase that increased dras- detected before formate in a batch phase when exposed to
tically after induction. This indicates higher respiration dur- increasing oxygen-limiting conditions. However, the con-
ing the production of the target protein and when growth centration of the latter increased first after the DOT showed
declines. a value of 0, which was never measured during the cultiva-
tions in the present investigation.
Glycolytic Byproducts Acetate and formate were produced in all cultivations, but
at different rates and at different times (Fig. 3). Generally,
Acetate is produced when E. coli is grown under anaerobic acetate is formed in the beginning of the process, later re-
or oxygen-limiting conditions (mixed acid fermentation) as placed by formate formation.
well as when growing aerobically in the presence of excess Acetate was accumulated during the batch and the expo-
glucose (overflow metabolism) (Lee, 1996). Formate, on nential feed phases. Acetate level was dependent on process

BYLUND ET AL.: SCALE-UP EFFECTS ON RECOMBINANT HUMAN GROWTH HORMONE PRODUCTION 123
process. However, the overall result of the SDR cultivations
gave the same byproduct pattern as for the other cultivations
(Fig. 4). 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. As can be seen
from Figure 4, the differences in biomass between cultiva-
tions cannot account for the differences in formate accumu-
lation.

Cell Lysis
The amount of DNA in the medium, which is comparable to
cell lysis, in relation to induction is shown in Figure 5.
These results show that the amount of DNA was highest in
scale-down cultivations, followed by pilot-scale experi-
ments, and was lowest in the control experiment. The ac-
cumulation rate increased after induction in all cultivations.
Notably, the pilot-scale experiment in which the recombi-
nant gene had been removed was similar to the lab-scale
control experiment, which shows that production of rhGH is
an additional source for cell lysis. The higher cell lysis in
scale-down experiments may partially explain the lower
biomass in these cultivations.

Product Formation
The total yield of product, including correct GH monomer
and its variants, is shown in Figure 6a. The general trend is

Figure 3. Formate and acetate accumulation during cultivations. (a)


Bench-scale control experiment, (b) scale-down experiments, and (c) pilot-
scale experiments. Repeated cultivations in the different reactors are num-
bered (1), (2), and (3) and marked with different symbols. Formate con-
centration marked as (1) , (2) , and (3) , respectively. Acetate con-
centration marked as (1) , (2) , and (3) , respectively. Pilot-scale
cultivation (3) corresponds to the experiment in which the recombinant
gene was removed from the plasmid.

time in the batch phase, where the higher the concentration


measured, the longer the period in the phase. The reason
why acetate was also produced during the late exponential
feed phase is probably due to the high specific growth rate.
It has been shown in the literature that overflow metabolism
occurs at substrate uptake rates comparable to a growth rate
of 0.30 to 0.35 h1 (in minimal medium), and to avoid
acetate formation the flux should be kept below this value
(Lee, 1996). Acetate was mainly consumed during the first
hours of the subsequent constant feeding phase.
Formate was produced after induction on a laboratory
scale. This byproduct accumulated rapidly up to a concen-
tration of 500 mg/L. In the SDR set-up, both metabolites
were produced in the PFR at all times (results not shown), Figure 4. Comparison of the different reactors with respect to (a) cell
growth shown as biomass (au), and (b) formate concentration (milligrams
indicating that acetate was more rapidly taken up by the
per liter). Cultivations marked as: control ; SDR ; and repeated pilot-
cells compared with formate, because acetate accumulated scale cultivations (1) , (2) , and (3) , respectively. Pilot-scale culti-
only in the beginning of the cultivation. 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.

124 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 69, NO. 2, JULY 20, 2000
Figure 5. DNA concentration in the medium (micrograms per milliliter).
The amount of DNA is correlated with the degree of cell lysis in the
experiments. Cultivations marked as: control ; SDR and ; and re-
peated pilot-scale cultivations (1) , (2) , and (3) , respectively. Pilot-
scale cultivation (3) corresponds to the experiment in which the recombi-
nant gene was removed from the plasmid.

a linear increase of product but at a lower level and rate in


the pilot-scale cultivations. This may be partially attribut-
able to the quality of the inoculum, and further points to the
difficulties of the scale-up procedure. The inoculum prepa-
ration is comparatively simpler in the laboratory, where
these experiments showed comparable results. The final
product mean values per cell dry weight in lab-scale (con-
trol and SDR) and pilot-scale experiments were 64.6 9.4,
75.8 11.1, and 41.7 8.2 au, respectively. The amount of
correct monomer (Fig. 6c) was, at the end of the cultiva-
tions, 70.0 1.8, 76.2 1.9, and 72.0 1.8% in the control,
scale-down, and pilot-scale experiments, respectively (val-
ues expressed with 95% confidence interval).
The amounts of proteolytically degraded forms of the Figure 6. (a) Total amount of rhGH per biomass (au) that includes cor-
product were different in the experiments. Figure 6b shows rect GH monomer and its variants; (b) amount of B (% of total GH),
that the amount of B, which includes LMW, clip-2, which includes LMW, clip-2, (Met(O)14)-rhGH, and deamidated rhGH;
and (c) amount of correct GH monomer (% of total GH). Cultivations are
(Met(O)14)-rhGH, and deamidated rhGH, was lowest in
marked: control, (1) ; SDR, (1) , (2) and (3) ; and pilot scale, (1)
scale-down experiments, with about 30% less B in the and (2) . Values are shown with 95% confidence interval.
final sample. The dominating form here was clip-2 and the
differences in the amount of trisulfide, for instance, were scale control experiments, respectively. The pilot-scale re-
smallonly about 1% higher during pilot-scale cultivations actor was mimicked in the SDR, but was exaggerated. No-
(result not shown). There were no apparent differences in tably, the amounts of formate were detected even though the
proteolysis between the laboratory-scale control and the pi- dissolved oxygen showed a value of 30% air in the stirred
lot-scale productions. tank reactor. Comparisons of protein production show that
the substrate oscillations decreased the accumulation of pro-
DISCUSSION teolytically degraded forms of rhGH.
Processes for recombinant protein production are often
The comparison of pilot-scale and scale-down two- difficult to control due to the number of different phases,
compartment experiments shows that the ruling regime on a such as growth phase (inoculum procedure, exponential and
pilot scale is mimicked in the SDR. This was concluded constant growth as well as declining growth), production
from the findings that the pattern of formate and DNA ac- phase (time of induction), and harvest. In the present inves-
cumulation could be simulated. However, there was a dif- tigation, the cultivations passed through different control
ference in the absolute value, where the highest values were regimes. Initially, variations in biomass concentration of the
found in the SDR, followed by the pilot-scale and the lab- inoculum were found to have a pronounced effect on a

BYLUND ET AL.: SCALE-UP EFFECTS ON RECOMBINANT HUMAN GROWTH HORMONE PRODUCTION 125
number of parameters, including biomass growth and ac- production of the recombinant protein had, in this specific
etate formation. This was seen in pilot-scale cultivations as case, a minor contribution to the decrease in yield coeffi-
well as on a laboratory scale. Before induction, the cultiva- cient.
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, but pected pattern for most experiments; that is, an increased
oxygen limitation due to scale effects was also clearly ob- concentration during the batch and exponential feed phase
served. At the point of induction, the increase in cell lysis followed by consumption during the constant feed (Fig. 3).
was severely interfering with the process performance, giv- The formation of acetate during the exponential feed phase
ing several effects: (i) oxygen limitation, although the DOT can, however, be avoided by choosing a specific growth rate
was >30% air saturation, which was seen from the increas- lower than 0.3 to 0.35 h1 (Lee, 1996). The acetate forma-
ing amounts of formate; and (ii) lower product formation tion pattern differed in control and one pilot-scale cultiva-
than expected. After this point the cultivations were tion compared with the others. In these cultivations, acetate
strongly influenced by the feed-controlled regime, because was not consumed at the same level during the early con-
any overfeed of glucose might result in a rapid accumula- stant feed. There was, however, a difference in that the
tion of the overflow metabolite acetate to lethal amounts, batch phase was longer in these experiments. The acetate
and limitation of oxygen because of the increased demand. was consumed immediately after induction and decrease in
Furthermore, during the production phase, several param- feed rate. 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.
value, although different depending on scale; increased pro- The most likely reason for the accumulation of formate
teolytical breakdown of the product; increasing mainte- seen in all cultivations is the appearance of local low oxy-
nance effects (reduced biomass); and a 100% increase in gen concentrations. This byproduct formation occurred de-
oxygen demand and carbon dioxide production per glucose spite the fact that dissolved oxygen tension was >30% air
added. The results point to the dynamics of recombinant saturation. 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. 4b) in which the for-
real-time scale. mate concentration increased first after induction. On the
Despite the fact that recombinant cultivations performed other hand, when the recombinant gene was removed from
on a pilot scale and in a control experiment showed a dif- the plasmid, the pilot-scale cultivation was similar to con-
ferent pattern with regard to glucose concentration, respira- trol experiments in this respect. This indicates that the de-
tion (YO2/S and YCO2/S), acetate, and formate accumulation, gree of oxygen limitation was influenced by both the induc-
similar biomass growth was generally obtained. The bio- tion as well as its scale dependence.
mass yield (YX/S) was 0.4 g/g in the cultivations during the The formate concentration in scale-down experiments
main part of the growth phase, but this value was succes- showed the highest value and the concentration also in-
sively decreased some hours before induction; therefore, the creased well before induction. These results clearly show
reduction took place mainly during the production phase. 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. 1). A similar maintenance effect has for substrates fed to the process. 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., 1998; Strandberg et al., 1994). The decrease al., 1998, 1999; George et al., 1998), and in this study was
in biomass yield was larger in the scale-down experiments, indicated by the variance of the glucose values in the pilot
resulting in a 6% to 10% lower final biomass. The reason reactor (Fig. 2). Furthermore, it has been shown in batch
for this was probably the stress introduced due to the fluc- experiments that the synthesis of specific proteins during a
tuations of glucose and oxygen concentrations and the fol- shift from aerobic conditions to anaerobiosis is in part simi-
lowing turn on/off of metabolism, which caused the forma- lar to the response of cells shifted to glucose starvation
tion of formate and also higher cell lysis (Figs. 3b and 5). (Nystrom, 1994). However, no increase in formate or ac-
However, this did not lead to increased degradation of prod- etate concentrations was obtained during glucose starvation
uct. The explanation why the biomass yield coefficient was conditions, even though the enzymes for the corresponding
lower in all the cultivations than the normal value for E. coli pathways were present. The rate of protein synthesis also
grown on glucose (0.5 g/g) is probably the high plasmid differed for a shift to anaerobiosis and glucose starvation
burden under uninduced conditions. It has been shown by conditions and was on the order of minutes to hours, re-
Seo and Bailey (1985) that carrying an increased number of spectively. Therefore, the low growth rate and the reduced
plasmid copies results in a decreased cell yield and specific feed rate in our experiments, which probably result in severe
growth rate. Furthermore, removing the recombinant gene glucose-limiting conditions, were most likely not the rea-
from the plasmid gave a similar low-yield coefficient of 0.4 sons for the production of formate.
g/g. After induction, the growth declined in this cultivation The amount of DNA in the medium, which is released
as in the others, but at a lower rate (Fig. 1c). Therefore, the due to cell lysis, showed a strong correlation with the ac-

126 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 69, NO. 2, JULY 20, 2000
cumulation of formate (Figs. 3 and 5). On the one hand, are more productive. However, this is at the expense of
oxygen limitation caused by the increased respiratory de- growth, because the cell mass is decreased and the cell lysis
mand might have caused the formate formation and cell is increased by the oscillations. The reason could be that
lysis, which in turn released DNA. On the other hand, the cells have a longer relaxation time when going from high to
presence of DNA due to induction could decrease the dif- low glucose concentration than vice versa. This means that
fusion rate of oxygen to the cell surface, thus causing an the cells imagine being in a richer environment for a
increased apparent saturation constant for this substrate. In longer time and hence no signals of severe limitation call for
any case, the results show that the concentration of oxygen degradation of high-energy internal products. An alternative
in the reactor can be rate limiting even at higher values; that is that the oxygen limitation following the high glucose
is, a higher DOT is needed to avoid mixed acid fermentation concentration reduces the amounts of otherwise formed pro-
products such as formate, acetate, ethanol, succinate, and teases that degrade the product. At first sight, the latter
lactate. seems more likely, because the greatest difference between
One may observe, in Figure 5, 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. However, it should be noted that, even though ac-
recombinant protein-producing cultivations. The most het- etate did not accumulate, it might have been formed but also
erogeneous environment is thought to be in the SDR, fol- consumed again when not exposed to zones with high-
lowed by the pilot-scale reactor and the homogeneous lab- glucose, low-oxygen concentrations.
scale reactor (control). However, it seems to be the higher
rate of release of DNA that causes the earlier accumulation CONCLUSIONS
of formate rather than its absolute value. This is since the
final formate concentration is similar in control and pilote- Precultivation stages must be reproducible to give reliable
scale experiments while the final DNA concentration dif- process results and thereby batch variations should most
fers. A reason why the DNA concentration is not propor- likely be avoided. 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. It is also critical to follow and control
the DNA change with cultivation time due to shear damage.
the substrate feed with the same accuracy in all scales.
During the process, 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 complex dynamics of
tween experiments. It was found that the amount of correct
recombinant processes, especially after induction, 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, low- oxy-
cose feed.
gen concentration in the two-compartment reactor. 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.e., the neous laboratory-scale and pilot-scale cultivations. How-
amount of B, with emphasis on clip-2). This cleavage site ever, 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, sub-
Tyr(143), which is an exposed domain easily accessible to strate uptake rate, production of acetate/formate and the
proteolytic enzymes (Gellerfors, 1990). It may therefore be accumulation interdependency, and cell lysis (i.e., a quali-
a connection between the degree of substrate gradients and tative resemblance). This was not found in absolute values.
proteolysis of a specific target protein when accumulated in But, there was a trend in the experiments, especially for
the periplasm. It is known that ATP-dependent proteolysis formate concentration and cell lysis, of increasing numbers
of staphylococcal protein A (SPA) is reduced when glucose from laboratory control, to pilot-scale, to SDR production.
concentration is kept low in the medium (Yang, 1995). This means that oxygen limitation periods are probably
However, most likely, growth hormone is not cleaved by an longer and/or relaxation times shorter in the SDR. A future
energy-dependent protease because the product is accumu- design might, therefore, be based on an SDR with a broader
lated in the periplasm. The proteolytic cleavage could, on residence time distribution.
the other hand, 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. The concentration of some recombinant performed all of the small-scale experiments.
proteins in batch cultivations has been shown to reach a
maximum value just before the glucose is exhausted and NOMENCLATURE
declines thereafter. This degradation is assumed to be due to
glucose starvation. Carbon-starved E. coli is, for instance, DOT dissolved oxygen tension (%)
OTR oxygen transfer rate (mol/Lh)
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., 1984). Therefore, the conclusion is that, in spite of the CSTR continuous stirred tank reactor
increased heterogeneity in the microenvironment, the cells vvm volume air per reactor volume and minute (L/Lmin)

BYLUND ET AL.: SCALE-UP EFFECTS ON RECOMBINANT HUMAN GROWTH HORMONE PRODUCTION 127
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