Impact of Autocrine Factors on Physiology and Productivity...
Transcript of Impact of Autocrine Factors on Physiology and Productivity...
Impact of Autocrine Factors on
Physiology and Productivity in
Trichoplusia ni Serum-Free Cultures
Ulrika ErikssonM. Sc.
Royal Institute of TechnologySchool of Biotechnology
Stockholm 2005
© Ulrika Eriksson
School of BiotechnologyDepartment of Bioprocess TechnologyRoyal Institute of TechnologySE-106 91 StockholmSweden
Stockholm 2005
ISBN 91-7178-056-4
Ulrika Eriksson (2005): Impact of Autocrine Factors on Physiology and Productivity inTrichoplusia ni Serum-Free Cultures. School of Biotechnology, Department of BioprocessTechnology, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.
Abstract
The aim of this study was to increase the understanding of the mechanisms regulating cellproliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T.ni) insect cells.
Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CMfactors) influencing cell growth. Metalloproteinase (MP) activity was the major factorresponsible for the growth stimulating effect of CM as shown by using the specific MPinhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due toautoproteolysis. Although the main band of the MP was determined to be around 48 kDa,precursor forms above 48 kDa as well as autocatalytic degradation products below the mainband could be observed. It is not clear whether all forms of the MP or just the main band isinvolved in the growth regulation. Further, a proteinase inhibitor could be identified in theinhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of anautocrine system regulating cell proliferation.
Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the wholeculture, indicating that S and G2/M are short relative to G1. After inoculation, a drasticdecrease in the S phase population together with a simultaneous increase of cells in G1 andG2/M could be observed as a lagphase on the growth curve and this may be interpreted as atemporary replication stop. When the cells were released from the initial arrest, the S phasepopulation gradually increased again. This was initiated earlier in CM-supplemented cultures,and agrees with the earlier increase in cell concentration. Thus, these data suggests acorrelation between CM factors and the cell cycle dynamics.
In cultures supplied with CM, a clear positive effect on specific productivity was observed,with a 30 % increase in per cell productivity. The specific productivity was also maintained ata high level much longer time than in fresh-medium cultures. The positive effect observedafter 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, theproductivity may be determined by the proliferation potential of the culture. A consequence ofthis would be that the secreted MP indirectly affects productivity.
Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which areessential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that innormal medium, while higher concentrations were inhibitory. However although vital, theywere not solely responsible for the growth-enhancing effect, as some other, more general,component present in yeast extract was needed for proliferation as well.
Keywords:Trichoplusia ni, High Five, insect cells, BEVS, serum-free medium, yeast extract, conditionedmedium, autocrine regulation of proliferation, growth factors, cell cycle, metalloproteinase,specific productivity
List of publications
This thesis is based on the following papers, which in the text will be referred to by
their Roman numerals:
I. Eriksson U, Hassel J, Lüllau E and Häggström L. (2005) Metalloproteinase
activity is the sole factor responsible for the growth-promoting effect of
conditioned medium in Trichoplusia ni insect cell cultures. (Submitted)
II. Calles K, Eriksson U and Häggström L. (2005) Effect of conditioned medium
factors on productivity and cell physiology in Trichoplusia ni insect cell
cultures. (Submitted)
III. Eriksson U and Häggström L. (2005) Yeast extract from Express Five SFM
contains essential factors for growth of Trichoplusia ni insect cells. (Submitted)
Aside from the presented papers, one additional paper has been written during the
course of this work.
• Svensson I, Calles K, Lindskog E, Henrikson H, Eriksson U and Häggström L.
(2005) Antimicrobial activity of conditioned medium fractions from
Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells. Appl.
Microbiol. Biotechnol. In press.
Contents
AIM OF STUDY........................................................................................................................................... 1
INTRODUCTION........................................................................................................................................ 2
INSECT CELL LINES .................................................................................................................................... 3
Present investigation............................................................................................................................. 4
CULTURE MEDIA ..................................................................................................................................... 5
SERUM-FREE MEDIA................................................................................................................................... 5
YEAST EXTRACT ........................................................................................................................................ 6
METABOLISM.......................................................................................................................................... 10
CARBOHYDRATES.................................................................................................................................... 11
AMINO ACIDS ........................................................................................................................................... 12
METABOLIC BY-PRODUCTS ..................................................................................................................... 13
CELL GROWTH AND REGULATION OF PROLIFERATION...................................................... 14
CELL CYCLE PHASE DISTRIBUTION AND GROWTH KINETICS ................................................................. 15
INSECT GROWTH FACTORS ...................................................................................................................... 19
CONDITIONED MEDIUM FACTORS............................................................................................................ 22
ORIGIN OF EXTRACELLULAR METALLOPROTEINASES............................................................................ 26
ROLE OF EXTRACELLULAR METALLOPROTEINASES IN REGULATION OF PROLIFERATION ................... 28
RECOMBINANT PROTEIN PRODUCTION ...................................................................................... 32
BACULOVIRUS EXPRESSION VECTOR SYSTEM........................................................................................ 32
PRODUCTIVITY – CONDITIONED MEDIUM FACTORS AND CELL CYCLE DYNAMICS .............................. 33
CONCLUSIONS AND FUTURE PERSPECTIVES............................................................................. 38
ACKNOWLEDGEMENTS ...................................................................................................................... 40
REFERENCES........................................................................................................................................... 41
1
Aim of study
Production of recombinant proteins is an important part of bioprocess technology of
today and the insect cell culture system has emerged as a competitive technology for
recombinant protein production. Some of the advantages are simplicity of cultivation,
high tolerance to metabolic by-products, high expression levels and the ability to
perform many of the post-translational modifications needed for biologically active
proteins.
Trichoplusia ni (T. ni) insect cells have gained increasingly popularity during the last
year as a possible host for protein production. However, the characteristics of this cell
line are not as well documented as that of Spodoptera frugiperda Sf9 insect cells.
Previous work has established that there is a difference in metabolism and physiology
between these two cell lines. The objective of this study was therefore to extend the
knowledge about the metabolism and physiology in T. ni serum-free cultures with the
long-term goal of improving growth and productivity.
The study was primarily focused on mechanisms regulating proliferation but also how
these mechanisms may affect recombinant protein production in the baculovirus
expression system. More specifically, the presence of an autocrine regulation system
was investigated and the significance of the alleged autocrine factors in regulation of
growth and productivity. The cell cycle dynamics were also studied as this is
intimately connected to the mechanisms controlling proliferation.
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Introduction
Developments in molecular biology and genetic engineering have had an enormous
impact on the biotechnology industry and the production of recombinant proteins plays
an increasingly important role. New techniques have made the discovery of thousands
of previously unknown genes possible and characterization of the corresponding
protein products require large amounts of proteins. In addition, the pharmaceutical
industry has great demands for high productivity whether the focus lies on screening
for a potential target or traditional production of a pharmaceutical protein product.
Depending on the purpose and protein to be produced, there are several different
expression systems available. The choice of expression system depends on the
intended use of the product, process goals and on the properties of the protein. Further,
productivity parameters in respect to yield and quality, as well as requirements for
post-translational modifications for biological activity are of importance. Prokaryotic
cells offer simplicity and large yields of product to low costs. Unicellular eukaryotes
as yeasts resemble mammalian cells in many respects but can be grown as easy as
prokaryotic cells, and to lower costs than mammalian cells. However, for production
of large and more complex proteins, the cellular environment of higher eukaryotes is
required. They are capable of performing many of the post-translational modifications
necessary for biologically active proteins. Indeed, there are advantages and
disadvantages of each system; however, this topic is beyond the work presented here
and described elsewhere (Fernandez and Hoeffler 1999).
Animal cell lines of mammalian origin have mainly been used for recombinant protein
production in the past (Koths 1995). However, insect cell systems have now emerged
in competition to mammalian expression systems mainly due to the increase in
diversity of applications with the baculovirus expression system (BEVS) (Kost and
Condreay 1999). Several other factors have also contributed to this popularity. Insect
cells are easy to cultivate and tolerate high concentrations of metabolic by-products. In
addition, high expression levels can be reached with relative ease and insect cells are
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also capable of many post-translational modifications (Beljelarskaya 2002). Hence, the
baculovirus-insect cell system is not only used in the expression of proteins for basic
research applications but also in the production of proteins valuable within the
pharmaceutical industry, such as therapeutic proteins and vaccines (DiFalco et al.
1997; Cruz et al. 1999; Guttieri et al. 2000).
Low volumetric productivity and high cost are the major disadvantages associated
with insect cells cultures. Runstadler (1992) suggested that the absolute most efficient
way to reduce the cost of a production process is to increase the specific productivity;
that is, to increase the amount of product produced per cell per unit time. During the
last decade, research and development focusing on increasing the cell productivity
have intensified (Hu and Aunins 1997). Several different factors influence cell growth
and recombinant protein production including bioreactor parameters, media
composition and cell specific characteristics, such as nutrient consumption rates,
accumulation of toxic by-products and the occurrence of autocrine factors (Runstadler
1992). This thesis will focus on the cell specific characteristics, and in particular on the
occurrence and effect of autocrine factors. The bioreactor parameters have been
extensively reviewed and will not be discussed here (Agathos 1996; Jäger 1996;
Ikonomou et al. 2003).
Insect cell lines
Grace established the first continuous insect cell line in 1962 by succeeding in
growing cells from the Antherea eucalypti female moth ovaries (Grace 1962). Since
then a variety of insect cell lines have been derived from different insect cell tissues
such as embryos, imaginal disks and ovaries (Baines 1996; Lynn 1996). Two
important cell lines for recombinant protein production are Spodoptera frugiperda Sf9
cells and Trichoplusia ni (T. ni, BTI-Tn5B1-4) cells, isolated from the ovaries of the
fall army worm and the cabbage looper, respectively (Vaughn et al. 1977; Granados et
al. 1994). The latter cell line is commercially known as the High Five cell line from
Invitrogen.
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Over 400 cell lines have been established so far from more than 100 insect species
(Lynn 1996), and it should be noted that cell lines originating from different insect
species tend to differ in their capacity to express recombinant proteins (Hink et al.
1991). T. ni insect cells have been proposed to be a superior host to many other insect
cells lines for recombinant protein production (Davis et al. 1993; Taticek et al. 2001).
For example, the expression level on a per cell basis has been suggested to be
considerably higher than that of Spodoptera frugiperda cells. Part of this increase in
expression is due to the fact that T. ni cells are 1.2-1.5 times larger than the
Spodoptera frugiperda cells. However, taking into account the differences in size, the
T. ni cells are still more productive on a per cell basis (Taticek et al. 2001). There are
also fundamental differences in cell physiology among the various cell lines, such as
different metabolism and growth rates (Öhman et al. 1996; Rhiel et al. 1997). These
disparities increase the demand for characterization of each individual insect cell line.
The different characteristics have to be taken under consideration before choosing an
optimal cell line for recombinant protein production, since they will affect cell growth
and the ability to produce the desired product. Important criteria to consider are growth
rate, ability to grow in suspension culture together with the ability to support synthesis
of recombinant proteins, post-translational modifications and secretion of the protein
product.
Present investigation
The data presented in this thesis is based on work performed on the T. ni cell line BTI-
Tn5B1-4 (High Five), and will hereon be refereed to as solely the T. ni insect cell line.
The medium used in this study is the commercially available serum-free medium
Express Five SFM (from Invitrogen). Some studies required yeast extract-free Express
Five SFM which was provided by the manufacturer.
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Culture media
One of the most important considerations in animal cell culture in general is the design
of an optimized medium which can support high density cell growth and high
production of recombinant proteins. The medium should supply a balance of
carbohydrates, salts, amino acids, vitamins, lipids and trace elements to meet the
nutritional needs of the insect cell lines. Initially, insect cells were cultured in basal
media supplemented with serum, in particular with a concentration of 5 to 20 % of
fetal bovine serum (FBS) (Grace 1962; Hink 1970). FBS has been shown to support
cell growth, baculovirus infection and recombinant protein production in insect cell
cultures (Wu et al. 1989). Suggested roles of serum are to supply growth factors and
nutrients (lipids, sterols, trace elements etc), hormones and to protect from biological
and mechanical damage (Maiorella et al. 1988). In spite of these advantages there are
also many drawbacks associated with the use of serum; high costs, uncharacterized
components, lot-to-lot variability in chemical composition are some of them. The use
of serum can also involve a risk for contamination by mycoplasma or virus. In
addition, the presence of serum in the culture medium complicates the downstream
purification process of the protein product and causes difficulties in metabolic studies
which require a chemically defined media. Further, production of pharmaceutical
protein products requires media without serum (Seamon 1998).
Serum-free media
Many of the above mentioned disadvantages are overcome by the use of a serum-free
medium and therefore, considerable work has been devoted to the development of
such media for insect cell cultures. The formulation of IPL-41 in 1981 provided a good
foundation for this development (Weiss et al. 1981) and IPL-41 has been used as a
base in several serum-free media (Maiorella et al. 1988; Ferrance et al. 1993;
Schlaeger et al. 1993). Although these serum-free media were optimized for
Spodoptera frugiperda cell lines, some of them support T. ni cell growth as well
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(Schlaeger 1996). However, due to different metabolism and higher nutrient demands
of the T. ni cell line (Yang et al. 1996; Rhiel et al. 1997; Taticek and Shuler 1997),
different composition of the medium might be required.
Medium development for T. ni insect cells resulted in the ISYL medium (Donaldson
and Shuler 1998a). This is a defined medium based on IPL-41 and supplemented with
protein hydrolysate, yeastolate, glucose and a lipid-pluronic emulsion. ISYL was
shown to support cell growth up to 6 x 106 cells ml-1 and it compared well with a
commercially available serum-free medium (Ex-Cell 405) in terms of volumetric
production of a recombinant protein product. Another approach was the use of a
factorial experimental design (Ikonomou et al. 2001). A variety of hydrolysates were
screened to determine which hydrolysate had the most important effect on cell growth.
Yeastolate ultrafiltrate was shown, by far, to have the most pronounced impact on cell
proliferation. This defined medium (YPR) supported good growth; T. ni cells reached
a final cell density of 6 x 106 cells ml-1 and was shown to be quite effective for the
production of recombinant proteins in comparison to the other media.
In addition to the contribution of different research groups, the industry has produced
an increasing number of commercially available serum-free media over the years.
Today, proprietary serum-free media are available which were developed specifically
for suspension cultures of certain insect cell lines, such as SF-900 II for the Sf9 and
Sf21 cell lines and Express Five SFM for T. ni cell line (both media from Invitrogen).
Yeast extract
Yeast extract products (or yeastolate) are produced by autolytic digestion of yeast; that
is, cell hydrolysis is performed by the endogenous proteinases of the yeast organism.
The resulting mixture contains amino acids, vitamins, especially the vitamin B
complex, nucleotides and other essential nutrients, and was developed as a nutritional
supplement for bacterial, insect and mammalian cell cultures. Some of the yeast
extract products are ultrafiltrated using a 10 kDa cut-off membrane to remove residual
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higher molecular weight contaminants, which could have proteolytic activity or
interfere with the purification process of the recombinant protein product. Yeast
extract has been used as a substitute for serum in serum-free media and was shown to
support cell growth to high densities (Drews et al. 1995; Donaldson and Shuler 1998a;
Ikonomou et al. 2001). Furthermore, addition of yeast extract promotes protein
production in insect cell cultures (Maiorella et al. 1988; Drews et al. 1995). Yeast
extract has also been used in different feeding strategies and shown to be very
effective for increasing protein productivity (Nguyen et al. 1993; Bédard et al. 1994;
Bédard et al. 1997). However, it is not fully understood which constituents of yeast
extract that are responsible for this enhancing effect. Although yeast extract is
considered to mainly be composed of amino acids, vitamins and nucleotides, it seems
as the growth promoting effect is due to other substances. Instead, yeast extract was
suggested to contain some smaller compounds that could act in a growth factor sense,
similar to that of serum (Wu and Lee 1998).
Results obtained in this study also confirmed the growth enhancing effects of yeast
extract (paper III). The origin of the yeast extract present in Express Five SFM is not
known since the composition of this medium is not disclosed by the manufacturer.
Nonetheless, it is most likely not ultrafiltrated through a 10 kDa cut-off filter since it
contained compounds in the molecular mass range of 10 to 25 kDa (see Fig. 2, paper
III). The observed compounds originated from the yeast extract since they were not
detectable in yeast extract-free medium. To study the effect of yeast extract on cell
growth, Express Five SFM was concentrated on a 3 kDa cut-off filter and fractionated
according to size on a gel filtration column. Important to notice is that the
concentration procedure does not enrich small molecules such as salts, vitamins and
amino acids, while larger compounds such as peptides and proteins may accumulate.
Fractions containing the yeast extract compounds were added to new cultures and were
shown to stimulate growth at concentrations up to 2.5-fold that in normal Express Five
SFM (Fig. 1). Higher concentrations were inhibitory to cellular growth. This was
supported by other experiments in which 10-fold concentrated Express Five SFM was
added directly to cultures and found to enhance growth (see Fig. 1, paper III). The
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observed concentration dependence of yeast extract on cell growth is in agreement
with earlier reports (Drews et al. 1995).
0
1
2
3
4
5
0 40 80 120 160
Via
ble
cell
den
sity
(10
6 c
ells
m1-1
)
Culture time (h)
Figure 1. Growth of T. ni cells in cultures supplemented with chromatographic fractions of Express Five SFM.Two added fractions are shown (, ) as compared to a reference culture in 100 % fresh medium (ο). Theconcentration of added yeast extract factors was estimated to be around 2.5-fold that in regular Express FiveSFM.
The factors found in the yeast extract present in Express Five SFM proved to be
important for T. ni cell proliferation, as it was not possible to remove them from the
culture medium and cultivate the cells in yeast extract-free Express Five SFM
supplemented with another type of yeast extract (Bacto Yeastolate) not containing
these compounds (Fig. 2). The first subculture grew normally, as would be expected in
yeast extract-containing Express Five SFM; however, further culturing of the cells led
to a drastic decrease in final cell concentration and after two passages proliferation
ceased. Nonetheless, it was possible to restore the capacity of the T. ni cells to grow in
this medium by supplementing chromatographic fractions with the yeast extract factors
present in Express Five SFM to the cultures (Fig. 3). Thus, these results supports the
fact that the factors present in the yeast extract used for preparation of Express Five
SFM are essential for T. ni cell growth.
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0
1
2
3
4
5
0 40 80 120 160 200 240 280
Via
ble
cell
dens
ity (
106 c
ells
ml-1
)
Culture time (h)
Figure 2. Successive cultures of T. ni cells in yeast extract-free Express Five SFM supplemented with BactoYeastolate. The inoculum for the first culture was taken from a pre-culture in Express Five SFM (containingyeast extract), but no spent medium was carried over. The four cultures represent passages 26 (), 27 (), 28 ()and 29 (ο), and were started from the previous culture on day three. The inoculum cell density was 3 x 105 cellsml-1.
0
1
2
3
4
5
6
7
0 50 100 150 200 250 300 350
Via
ble
cell
dens
ity (
106 c
ells
ml-1
)
Culture time (h)
Figure 3. Effect of added yeast extract factors from Express Five SFM to a culture with yeast extract-freeExpress Five SFM supplemented with Bacto Yeastolate. The yeast extract factors were present from thebeginning of culture. The inoculum for the first culture was taken from a pre-culture in Express Five SFM(containing yeast extract), but no spent medium was carried over. Four subcultures are shown: passages 27 (),28 (), 29 () and 30 (ο). The last three cultures were started from the previous culture on day three. Theinoculum cell density was 3 x 105 cells ml-1.
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However, the function of yeast extract in insect cell cultures seems to be even more
complex. The cells could not grow in yeast extract-free Express Five SFM
supplemented with the chromatographic fractions alone. Hence, these results indicate
that some other components in yeast extract are absolutely necessary for proliferation.
This is not unexpected, since yeast extract is considered to substitute many functions
of serum. These components are most likely peptides or other small molecules since
ultrafiltrated yeast extract also promotes cell growth (Maiorella et al. 1988; Ikonomou
et al. 2001). One should bear in mind though, that the quality of yeast extract varies
depending on the origin and the handling procedure and this may lead to lot-to-lot
variability with different responses in respect to cellular performance.
Although yeast extract is a key component in serum-free media as a source of essential
nutrients, it is clear that yeast extract contains undefined components which may have
a large impact on cell behavior. As previously discussed, metabolic studies benefit a
great deal from using a chemically defined medium and therefore, attempts to
substitute yeast extract with chemically defined substances is of importance. Wilkie et
al. (1980) claimed to have developed such a chemically defined media lacking yeast
extract, which could support growth of Spodoptera frugiperda cells. However, several
attempts by other research groups to use this medium proved unsuccessful, and
culturing of the cells became possible only when the medium was supplemented with
yeast extract (Ferrance et al. 1993; Bhatia et al. 1997).
Metabolism
Several studies deal with the metabolism, physiology and the nutritional requirements
of insect cells during serum-free conditions. However, most of this work has been
conducted on the Spodoptera frugiperda Sf9 cell line, and much less has been made
with the T. ni cell line. Since these cell lines differ in metabolism and physiology,
individual characterization of the cell lines are important.
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Carbohydrates
Different insect cell lines exhibit different patterns of carbohydrate utilization.
Stockdale and Gardiner (1976) studied the utilization of 21 sugars by T. ni and
concluded that only five supported cell growth, namely glucose, fructose, mannose,
maltose and trehalose. Glucose and trehalose were consumed at a higher rate than
fructose. Sf9 cells was also shown to prefer glucose to fructose (Bédard et al. 1993);
thus, glucose is now considered to be the most important carbohydrate sources for
insect cell cultures. As T. ni cells have been suggested to have a higher metabolic
activity than Sf9 cells, it is not surprising that the utilization rate of glucose was shown
to be significantly higher for T. ni cells (Rhiel et al. 1997). Further, the specific
glucose consumption rate for T. ni cells varied between different media, indicating that
the nutritional requirements for the same cell line may differ depending on which
medium that is used. However, the maximum cell density was reached prior to glucose
exhaustion. These results are in agreement with our data obtained in Express Five
SFM (Fig. 4). Further, Rhiel et al. (1997) showed that T. ni cells continued to consume
glucose at a high rate during the transition from exponential to stationary growth and
during the stationary phase until complete exhaustion. Glucose did not become
limiting in our cultures; however, glucose exhaustion during the stationary growth
phase can not be excluded since no samples were taken.
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0
20
40
60
0
2
4
6
0 40 80 120 160
Glu
cose
con
cent
ratio
n (m
M)
Viable cell density (10 6 cells m
l -1)Culture time (h)
Figure 4. Glucose concentration profile () in a T. ni cell culture in Express Five SFM, shown together with theviable cell density (ο).
Amino acids
Amino acids are important in the biosynthesis of proteins and for energy production;
however, insect cells are not able to synthesize most of the amino acids themselves
(Bhatia et al. 1997). For T. ni cells, the most important amino acids are asparagine,
glutamine, and cystine. Asparagine is rapidly consumed in T. ni cell cultures and is the
first amino acid to become exhausted, followed by glutamine and cystine (Yang et al.
1996; Rhiel et al. 1997). Yang et al. (1996) also showed that while asparagine
depletion coincided with the onset of the stationary phase, glutamine depletion did not.
Thus, T. ni cells preferentially utilized asparagine before glutamine (Rhiel et al. 1997).
In our cultures, asparagine was rapidly consumed and depleted after 96 h and was,
together with cystine, the first limiting amino acid (Fig. 5a). The entry into the
stationary phase occurred about the same time as asparagine depletion, as noted by
Yang et al. (1996). In addition, glutamine was also utilized at a high rate but was not
exhausted during culture (Fig. 5a). None of the other amino acids were consumed to
any great extent; by the end of the culture the concentrations of all other amino acids
remained at > 50% of their initial values.
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0
5
10
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20
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0.5
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0 40 80 120
Asp
arag
ine
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amin
e co
ncen
tratio
ns
(mM
)
Cystine concentration (mM
)
Culture time (h)
a
0
5
10
15
20
0 40 80 120
By-p
rodu
ct c
once
ntra
tions
(m
M)
Culture time (h)
b
Figure 5. Consumption of amino acids and accumulation of by-products during a T. ni cell culture in ExpressFive SFM. (a) The amino acid utilization is shown for asparagine (), glutamine (ο) and cystine (). (b)Production of the metabolic by-products ammonium (), lactate (ο) and alanine ().
Metabolic by-products
T. ni cells accumulate lactate to rather high concentrations, resembling the metabolism
of mammalian cells (Rhiel et al. 1997). The medium composition seems to be
important for the amount of lactate produced, as demonstrated by Rhiel et al. (1997).
Considerably more lactate was produced in a medium which was not optimized for T.
ni cells than in a medium specifically designed for this particular cell line (Express
Five SFM). In our cultures, the amount of lactate produced in Express Five SFM was
less then 10 mM (Fig. 5b), which is comparable with the data by Rhiel et al. (1997). In
addition, T. ni cells produce large amounts of ammonium during culture,
concentrations up to 35 mM have been reported (Rhiel et al. 1997). Although insect
cells are not as sensitive as mammalian cells to the presence of ammonium,
concentrations above 25 mM have been shown to severely impair recombinant protein
production while concentrations lower than 15 mM had no effect (Yang et al. 1996;
Chico and Jäger 2000). Ammonium accumulated up to 20 mM in our T. ni cultures in
Express Five SFM, together with an alanine production up to 15 mM. The amount of
by-products produced are not thought to be inhibitory to cell growth or affect
recombinant protein production in a negative way (will be further discussed in the
section “Productivity – Conditioned medium factors and cell cycle dynamics”).
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Further, ammonium and alanine are generated by the asparagine and glutamine
metabolism, while lacate is derived from glucose. However, not so much is known
about the metabolism of T. ni cells and only a few reports regarding this matter have
appeared in the last decade (Ikonmou et al. 2003).
Cell growth and regulation of proliferation
Proliferation of insect cells is characterized by a lagphase upon inoculation to fresh
medium, and this lagphase is a cellular response to new culture conditions. The initial
seeding cell density is one parameter affecting the lagphase, as a lower inoculum cell
concentration typically results in a longer lagphase than a higher inoculum cell
concentration does. This is particularly true during serum-free conditions, and has
previously been observed for both Sf9 (Hensler et al. 1994; Doverskog et al. 2000) and
T. ni insect cells (Taticek et al. 2001). The choice of inoculum cell concentration
influences the overall cell growth as well, with higher final cell densities being reached
with higher seeding densities. When the inoculum cell density was varied in the range
of 1.5 to 3.0 x 105 cells ml-1, higher maximum cell densities and shorter lagphases
were obtained in cultures inoculated at higher cell concentrations (see Fig. 1, paper I).
Further, cultures inoculated at cell concentrations between 0.3 x and 3.0 x 106 also
confirmed the correlation between initial seeding cell density and lagphase length (Fig.
8a). However, the effect on maximum cell concentrations was not investigated in this
experiment since the cultures were only followed for the first 48 hours.
The relationship between lagphase, inoculum cell density and cellular performance is
recognized from cell lines secreting autocrine growth factors into the culture medium
(Lauffenburger and Cozens 1989). The hypothesis is that a certain time period is
required for the critical factor(s) to accumulate to sufficient amounts necessary for
initiation of growth, and at lower cell concentrations this takes a longer time, resulting
in a longer lagphase. Moreover, proliferation of Sf9 insect cells in serum-free cultures
has been proposed to be a result of auto-regulatory events controlling both growth and
metabolism, and particularly secreted autocrine factors were suggested to play an
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important part of regulation (Doverskog et al. 1998; Doverskog et al. 2000). This
suggestion appears reasonable since cloned insect cell lines are derived from various
insect tissues, and thus, these cell lines may also require mitogenic stimulation by
external factors to be able to proliferate in culture. Although considerable research
efforts have been devoted to characterize insect growth factors in general, much less is
known about the occurrence of autocrine growth factors and their significance in
cloned insect cell lines. Therefore, one aim of this work was to investigate the
presence of autocrine factors in serum-free cultures of T. ni cells and the significance
in growth regulation (paper I).
Cell cycle phase distribution and growth kinetics
The cell cycle dynamics of a culture is closely connected to cell growth and to the
mechanism controlling proliferation, so before discussing insect growth factors and the
possibility of regulation by an autocrine system, a discussion of the cell cycle
dynamics during serum-free conditions would be appropriate.
Proliferating eukaryotic cells proceed through an ordered set of events termed the cell
cycle. As illustrated in figure 6, the major steps in the cell cycle are the G1 (G denotes
gap), S, G2, and M phases. The S (synthesis) phase is the period of DNA synthesis,
and the M (mitosis) phase is when cell division occurs. The G1 and G2 phases
separates the replication and the mitosis events. The passage of a cell through the cell
cycle is highly regulated at checkpoints, where key decisions are made. The first of
them in the late G1 phase is the restriction point which commits the cells to initiate
DNA synthesis. The other two checkpoints are the entry and the exit of mitosis
(Fussenegger and Bailey 1998). All of these checkpoints are tightly controlled by
proteins in the cytoplasm; especially by the cyclin dependent kinases and the cyclin
proteins (Coleman and Dunphy 1994; Morgan 1995). Normal cells require external
signals to proceed through the cell cycle, even in the presence of essential nutrients.
The absence of appropriate growth factors or unsuitable environmental conditions send
cells into a non-growing state, the G0 phase. Cells in G0 can be stimulated to
16
proliferate again in the presence of appropriate extracellular signals (Fussenegger and
Bailey 1998).
Figure 6. Overview of the eukaryotic cell cycle.(http://www.emc.maricopa.edu/faculty/farabee/BIOBK/cellcycle.gif)
The duration of the cell cycle varies among different cell types, for animal cells it is
normally between 10 to 30 h long. Also, the relative length of the various phases in the
cell cycle may differ depending on local conditions. Mammalian cells have a high
percentage of cells in the G1 phase during culture (Fertig et al. 1990) in contrast to Sf9
insect cells, which have been suggested to have a high fraction of cells in the G2/M
phase instead (Fertig et al. 1990; Doverskog et al. 2000). However, results from our
experiments proposed that there is a difference in cell cycle dynamics between insect
cell lines as the cell cycle kinetics of T. ni cells more seems to resemble that of
mammalian cells (paper II).
The majority of the cells, approximately 80 to 90 %, was found in the G1 phase
throughout the whole culture. The remaining 10 to 20 % were found in the S or G2/M
phases (Fig. 7a). This uneven distribution indicates that the S and G2/M phases are
short relative to the G1 phase of the cell cycle. This conclusion agrees with the
estimation by Lynn and Hink (1978a) that the S phase was only about four hours in T.
ni cells. Further, a dramatic decrease of cells in the S phase could be observed upon
inoculation of cells into fresh serum-free medium, together with a simultaneous
increase of cells in G1 and G2/M phases. The decrease of the number of cells in S
17
could be observed as a lagphase on the growth curve, and may be interpreted as a
temporary replication stop. After a few hours, the S phase population gradually
increased again and remained at the highest level for about 24 h, which was followed
by a decline in the amount of cells present in this phase. This was accompanied by an
small increase of cells into the G1 phase (Fig. 7a). This observed G1 accumulation late
in culture, as well as the initial G1 accumulation, coincided with an abrupt increase in
cell diameter during these phases (Fig. 7b). The sharp upward turn of the cell size
profile after 80 hours likely reflects the onset of down-regulation of proliferation, as
described for Sf9 cells (Doverskog et al. 2000). Thus, cell diameter (or cell volume) is
an important parameter reflecting cell physiology, as it is related to changes in the cell
cycle dynamics and the cells’ metabolic state (Ramírez and Mutharasan 1990). The
measured cell diameter is a mean value for the whole population, which in this case
mainly reflects the size of the large G1 population.
0
5
10
15
20
25
50
60
70
80
90
0 40 80 120
Cell
cycl
e di
strib
utio
nS,
G2/
M (%
)
Cell cycle distribution G1 (%
)
Culture time (h)
a
18
19
20
21
0 40 80 120
Cell
diam
eter
(µm
)
Culture time (h)
b
Figure 7. (a) Distribution of the cell population in G1 (ο), S () and G2/M () phases of the cell cycle, and (b)variations in cell diameter in T. ni cultures.
Different seeding cell concentrations also had a large impact on the cell cycle
distribution. Cultures were inoculated to cell densities between 0.3 x 106 and 3 x 106
cells ml-1 in fresh medium and followed for the first 48 h. Cultures inoculated at the
18
two largest inoculum sizes (2 and 3 x 106 cells ml-1) exhibited a much shorter lagphase
as compared to cultures with lower seeding cell densities, and they reached the
maximum cell concentration within the time period studied (Fig. 8a). As previously,
the majority of the cells were found in the G1 phase. However, the most remarkable
result was the difference in the S phase dynamics between the various cultures (Fig.
8b). The initial decrease of cells in the S phase occurred at about the same rate in all
cultures, but the increase of S phase cell population was initiated much sooner in
cultures inoculated at higher cell concentrations. This was most evident when
comparing the highest inoculum cell concentration to the lowest. Further, for the
highest seeding cell densities the S phase population immediately decreased after
reaching its maximum level. These results may be interpreted as the high inoculum
cultures were more synchronized than those at low inoculum cell densities, but, on the
other hand, that proliferation was down-regulated much sooner in the high inoculum
cultures.
0
2
4
6
0 10 20 30 40 50
Via
ble
cell
dens
ity (
106 c
ells
ml-1
)
Culture time (h)
a
0
2
4
6
8
10
12
14
0 10 20 30 40 50
Cell
cycl
e di
strib
utio
n S
(%)
Culture time (h)
b
Figure 8. (a) The influence of inoculation cell concentrations on lagphase and viable cell density. Seedingdensities: 0.3 x 106 (ο), 0.5 x 106 (), 0.7 x 106 (), 1.0 x 106 (), 2.0 x 106 () and 3.0 x 106 () cells ml-1. (b)The difference in S phase dynamics between cultures inoculated at 0.3 x 106 (ο), 0.7 x 106 (), 1.0 x 106 () and3.0 x 106 () cells ml-1. The other two cultures fell in between these cultures.
19
Insect growth factors
All normal cells are highly regulated through the action of different growth factors
such as proteins and hormones. Important functions of growth factors are to regulate
cell growth and division and to control survival, differentiation and migration of cells.
Most actions are mediated by a specific combination of growth factors rather than by
a single growth factor (Alberts et al. 1994). Within the insect, hormones are important
in every aspect of development and reproduction, and control a variety of
physiological, developmental and behavioral phenomena (Nijhout 1994).
The main classes of insect hormones are those secreted by the endocrine glands, the
ecdysteriods and the juvenile hormones, and those produced by the neurosecretory
cells in the brain, the neurohormones (Gade et al. 1997). The first hormone to be
discovered in insects was the polypeptide neurosecretory prothoracicotropic hormone
(PTTH), which induces the production of ecdysteroids in the prothoracic glands.
Ecdysteroids are together with the juvenile hormones, the two principle hormones in
insects that coordinate growth and development. They are important in the regulation
of molting and metamorphosis and play a great role in almost every aspect of an
insect’s life. In the majority of insects, the prothoracic glands are the major source for
ecdysteroids; however, the ovary has been shown to be a source for this hormone as
well (Nijhout 1994). Some insect cell lines are known to be sensitive to ecdysteroids
applied at a physiological level. The responses include morphological changes such as
cell elongation and changes in cell volume (English et al. 1984; Cassier et al. 1991) as
well as biochemical changes such as the synthesis of specific extracellular proteins,
proteoglycans (Porcheron et al. 1991). In addition to morphological and biochemical
effects, ecdysteroids have also been suggested to have an impact on protein production
and cell growth. For example, addition of ecdysteroids to Sf9 insect cell cultures
increased the yield of recombinant proteins produced (Sárvári et al. 1990; Chan et al.
2002), while an inhibitory effect was observed on cell proliferation in an epidermal
lepidopteran cell line (Hatt et al. 1997; Auzoux-Bordenave et al. 2002).
20
Another well-characterized neuropeptide is the neurohormone bombyxin (previously
termed the small prothoracicotropic hormone, PTTH-S), isolated from the insect
species Bombyx mori. Even though this hormone was originally classed as a
neuropeptide, tissues such as the epidermis, testis, ovary and fat body produce
bombyxin as well (Iwami et al. 1996a). Specific bombyxin receptors have also been
reported and characterized for a variety of insect species (Fullbright et al. 1997).
However, the biological function of bombyxin is not fully understood. It has been
suggested to be involved in the reproduction process due to the presence of specific
bombyxin receptors on the ovaries (Fullbright et al. 1997). Further, the structural
similarities of bombyxin to insulin and insulin-like growth factors have also led some
authors to suggest that bombyxin may play a role in the growth regulation (Iwami et
al. 1996b). For example, bombyxin has been suggested to stimulate cell division and
growth in wing imaginal disks in lepidopteran insects, by acting together with
ecdysteroids (Nijhout and Grunert 2002). In addition to bombyxin, several other
insulin-like peptides have been identified in a variety of insect species (Kramer 1985),
and some of them have also been proposed to be involved in the regulation of growth
(Brogiolo et al. 2001; Krieger et al. 2004).
Over the last 15 years, several other peptides with growth stimulatory activities have
been identified in insects, such as sapecin and the growth-blocking peptide (GBP)
(Komano et al. 1991; Hayakawa and Ohnishi 1998). Sapecin is known to have potent
antibacterial activity but was also suggested to stimulate embryonic cell proliferation.
GBP is an insect cytokine, which, in addition to controlling the larval development of
lepidopteran insects, also acts as a mitogen for various types of cultured cells. Another
peptide with a growth factor function is the Bombyx mori paralytic peptide (BmPP),
which promoted cell growth in a number of different insect cell lines (Sasagawa et al.
2001). Further, the first polypeptide growth factors to be reported from insects were a
family of Imaginal Disc Growth Factors (IDGF) from Drosophila (Kawamura et al.
1999). IDGFs work together with insulin to stimulate cell growth of cultured imaginal
disk cells.
21
Vertebrate growth factors supplied to various insect cell cultures largely failed to
support cell growth (Ferkovich and Oberlander 1991; Nishino and Mitsuhashi 1995).
However, the use of vertebrate insulin has shown ambiguous results. For example,
insulin promoted growth and could completely replace serum in Drosophila cell
cultures (Davis and Shearn 1977; Mosna 1981). On the other hand, insulin could not
be used to stimulate cell growth in lepidopteran cell cultures (Hatt et al. 1997), or be
used as a substitute for serum in a Mamestra brassicae cell culture (Nishino and
Mitsuhashi 1995). Further, addition of insulin to our T. ni cultures did not have a
stimulatory effect on cell growth. On the contrary, the results rather suggested a
negative influence on cell proliferation in a dose-dependent manner (Fig. 9).
0
2
4
6
0 40 80 120
Via
ble
cell
dens
ity (
106 c
ells
ml-1
)
Culture time (h)
Figure 9. The effect of different concentrations of insulin on T. ni cell proliferation. The added concentrationswere 5 mg l-1 () and 10 mg l-1 () in comparison to a reference culture without insulin (ο)
These large variations in response to added insulin propose diverse regulatory
mechanisms acting in different insect cell lines. The presence of the insects own
insulin-like peptides may also interfere with the regulation mechanism, such as the
presence of the insulin-like peptide binding proteins (IGFBP) in Sf9 and T. ni cultures
(Doverskog et al. 1999; Andersen et al. 2000). Interestingly, the amount of a protein at
27 kDa, as estimated by silver-stained SDS-PAGE, increased in the insulin-
22
supplemented cultures. This protein has not been identified, but it appeared at the same
molecular mass as the IGFBP previously identified in T. ni cultures (27 kDa). Hence,
since these IGFBPs are known to bind insulin, it is possible that the added insulin
stimulate the production of the IGFBP.
Conditioned medium factors
Cultured Sf9 cells have been proposed to produce factors which are important in the
regulation of proliferation (Doverskog et al. 2000). The presence of such factors was
indirectly demonstrated through the effects of conditioned medium (CM) on cell
growth and proliferation. Addition of CM stimulated proliferation by shortening the
lagphase and increasing the final cell concentration. Thus, they concluded that CM
contained mitogenic factors which stimulated proliferation. Support for a similar
regulation system in T. ni cell cultures was obtained in several experiments with CM
from our T. ni serum-free cultures (paper I).
Addition of 20 % CM decreased the length of the lagphase and increased the
maximum cell concentration (Fig. 10a). The growth enhancing effect of CM was
dependent on the concentration. The initial lagphase was affected by all
concentrations tested (10, 20, and 30 %); however, the maximum cell density was only
influenced by 20 % CM (Fig. 10a). The effect also depended on seeding cell
concentrations, and became more pronounced at lower inoculum cell densities (see
Fig. 1, paper I). Further, CM harvested at days two and three of a previous culture
stimulated cell growth in concentrations of 20 %, while 20 % CM from day one did
not (see Fig. 2, paper I). However, this was a concentration issue as 100 % CM from
day one could support a better growth. Proliferation in CM harvested at later time-
points seemed contradictory, as demonstrated by different cellular responses. The cells
were stimulated by CM from days four and five in some experiments while not in
others. What is causing these different responses remains unclear. T. ni cells used in
this study are between passages 15 to 30 which means that new cultures were started
from new ampoules every other month. An observation was that the cells behaved
23
differently from time to time, even if they originally stemmed from the same master
stock. One explanation might be that handling of the cells over time results in a
selection of a certain type of cells.
0
1
2
3
4
5
0 40 80 120 160
Via
ble
cell
dens
ity (1
06 cel
ls m
l-1)
Culture time (h)
a
18
19
20
21
0 40 80 120
Cell
diam
eter
(µm
)
Culture time (h)
b
Figure 10. (a) The influence on T. ni cell growth by addition of 10 % (), 20 % () and 30 % () CM, ascompared to 100 % fresh medium (Ο). (b) The effect of 20 % CM () on cell diameter as compared to areference culture (Ο). CM was raised from a previous culture on day three and the inoculum cell density was 1.5x 105 cells ml-1. The data on cell growth and cell diameter are from different experiments.
Interestingly, cell populations exposed to CM were smaller in diameter when
compared to a reference culture, with a 16 % size difference at the maximum cell
volume (Fig. 10b). Cell cultures supplemented with CM remained smaller throughout
the growth phase, but reached the same size as the reference cell population at the end
of the culture. The difference in size was most evident in the beginning of the culture
where CM stimulated proliferation. As the measured cell diameter is a mean value for
the whole cell population and the G1 phase is long in T. ni cell cultures, the G1 cells
should be rather heterogeneous in size. Thus, the CM-culture contains a larger
proportion of newly, divided small G1 cells, whereas the larger cells in the reference
culture may represent a majority of late G1 cells, waiting to enter the S phase.
24
In contrast to the effect on diameter, cell cycle analysis of CM-supplemented cell
cultures showed no apparent change in the overall cell cycle distribution (see Fig. 2,
paper II). However, addition of CM led to an earlier increase of the number of cells in
the S phase suggesting an earlier release from the temporary replication stop and entry
into the exponential growth phase. This result agrees with the earlier increase in cell
concentration in CM-containing cultures than in fresh medium cultures (Fig. 10a).
Hence, we propose that there might be a correlation between CM factors and changes
in the cell cycle dynamics. This has previously been proposed to be the case for
cultured Sf9 cells; that is, cell cycle progression is a result of auto-regulatory events
occurring at key-points during culture (Doverskog et al. 2000).
The components in CM were fractionated according to size on a gel filtration column.
SDS-PAGE analysis of the chromatographic fractions revealed a large amount of
extracellular proteins produced by the T. ni insect cells during serum-free conditions
(see Fig 3, paper I). As the yeast extract used in Express Five SFM had been shown to
contain some factors influencing growth, yeast extract-free medium was used to ensure
that the proteins unquestionably originated from the cells and not from the medium.
Although T. ni cells do not proliferate in yeast extract-free medium, CM from these
cultures had the same growth-promoting effect as CM from normal medium.
Supplementation of new T. ni cultures with the individual chromatographic fractions
showed that fractions eluting at around 45 kDa had a significant stimulatory effect on
cell proliferation, while fractions with lower molecular mass components (10 to 15
kDa) exhibited a clear negative effect on cell growth (see Figs. 4 and 5, paper I). No
other fractions tested had an obvious effect on proliferation. Thus, these results
concluded that CM from T. ni insect cells contains at least one growth-promoting
factor in the molecular mass range of 45 kDa and an inhibitory factor of a lower
molecular mass.
Attempts to identify the proteins responsible for these observed effects on proliferation
were made by subjecting proteins of interest to N-terminal amino acid sequencing. A
protein with a molecular mass of 43 kDa was selected as probably being responsible
25
for the growth-stimulatory effect (indicated by an arrow in Fig. 11a). This protein band
was selected by comparing the protein pattern in the stimulatory fractions with that of
adjacent fractions without effects. The 43 kDa factor was found to be 67 % identical to
a sequence of a snake venom metalloproteinase; however, the sequence of the 43 kDa
factor was not found in the N-terminus but rather at a position starting at amino acid
174. This result is not unlikely since most extracellular metalloproteinases (MPs) are
secreted as inactive precursor, containing a pro-peptide in the N-terminus, which is
cleaved off upon activation (Nagase 1997). Possible role of secreted MPs in growth
regulation will be further discussed in the section “Role of extracellular
metalloproteinases in regulation of proliferation”.
In the inhibitory fraction, three out of the four protein bands visible on a silver-stained
gel could be identified (Fig. 11b). The database search indicated that the fraction
contained a probable cyclophilin, a lysozyme precursor and a kazal-type proteinase
inhibitor (see Table 1, paper I). The fourth band could not be identified due to
blockage of the N-terminus. To mention a few facts about these proteins; cyclophilins
have mainly been ascribed intracellular functions; however, recent reports suggest a
functional role for secreted cyclophilins as well (Jin et al. 2000). Further, the lysozyme
precursor protein is assumed to have antimicrobial activity and have previously been
identified in the T. ni larvae as well (Kang et al. 1996). Finally, although the kazal-
type proteinase inhibitors are most frequently involved in the inhibition of serine
proteinases, kazal-type modules may be part of larger multidomain proteinase
inhibitors which control other classes of proteinases as well (Trexler et al. 2001). The
cyclophilin and lysozyme proteins are not though to be responsible for the inhibitory
effect, while the role of the secreted proteinase inhibitor remains to be investigated.
26
Figure 11. Protein composition in the fractions with an influence on cell proliferation, as visualized by silver-stained SDS-PAGE. (a) Growth-stimulatory fractions; the arrow indicates the 43 kDa protein band selected forN-terminal amino acid sequence analysis. (b) Growth-inhibitory fraction; all of the observed protein bands weresubjected to N-terminal amino acid sequencing. The protein bands correspond to: 1 cyclophilin (22.5 kDa), 2lysozyme precursor (16.3 kDa) and 4 proteinase inhibitor (10.5 kDa). Band no 3 could not be analyzed due to N-terminal blockage.
Origin of extracellular metalloproteinases
Ikonomou et al. (2002) have previously described the presence of two secreted MPs,
of which one was in the same molecular mass range (42-43 kDa) as the sequenced
proteinase in this study. To verify the presence of MP activity in our cultures, casein
zymographic gels were run with samples from both CM and from the fractions with a
growth-promoting effect. Several proteinase bands were detected within a molecular
mass range of 32 to 73 kDa, and the molecular mass of the main band was determined
to be around 48 kDa. Although several bands could be observed in both non-
concentrated and concentrated CM, bands below and above the main band were much
more pronounced in concentrated samples (see Fig. 6, paper I). Further, complete
inhibition of the activities was obtained with the MP inhibitors EDTA and DL-
thiorphan, showing that the observed proteinase bands indeed were MPs.
Extracellular MPs are known to exist in several different molecular mass forms due to
degradation by autoproteolysis. Many MPs are also synthesized and secreted from the
cells as inactive proenzymes which are activated extracellularly in a stepwise manner
(Nagase 1997). Therefore, our data suggest that the observed proteinase bands are not
different enzymes but precursor form and degradation products of the same enzyme.
The activation of the precursor form is often initiated by already active enzymes, but
27
SDS, urea or other denaturants may also activate the precursor which allows the
proenzyme to be detected by zymography (Kleiner and Stetler-Stevenson 1994).
Hence, it is possible to detect both precursor forms and active forms of proteinases on
zymographic gels.
Support for the proposition that the observed proteinase was just one instead of several
different enzymes was obtained from experiments with CM and EDTA. Concentration
of CM without EDTA induced the appearance of several proteinase bands below the
main band of 48 kDa whereas, addition of EDTA suppressed the emergence of most of
the additional bands (Fig. 12). Thus, these results indicate that the bands below the
main band were formed by autocatalytic mechanisms and that EDTA inhibited this
autocatalytic activity of the MPs present from the beginning in the sample, thereby
preventing further degradation during the concentration procedure. The activity of
MPs are highly regulated through different mechanisms and autoproteolytic
inactivation is one of them. For instance, brevilysin H6 (a snake venom
metalloproteinase) may be autocatalytically cleaved at more than 25 different amino
acid residues (Fujimura et al. 2000). Some of these cleavages inactivate the MPs,
while other generate truncated enzymes with the ability to cleave some substrates but
lose the ability to cleave others (Woessner and Nagase 2000). Clearly, the main
truncated forms of the T. ni MP (43, 32 and 25 kDa) retain casein-degrading activity
since they are visible on the zymography gel (Fig. 12 and see Fig. 6, paper I). As
previously discussed, Ikonomou et al. (2002) reported on two MPs at 33 and 42 kDa
respectively, and our data propose that these MPs correspond to the degradation
products reported here.
28
Figure 12. Zymography analysis of CM supplemented with 5 mM EDTA during the concentration procedure, ascompared to concentration in the absence of EDTA. CM was concentrated 5-fold on a 10 kDa cut-off filter. Non-concentrated CM (without EDTA treatment) was used, to show that the concentration procedure itself inducedthe appearance of additional proteinase bands. Samples were taken from days three and four of a T. ni culture.Lanes 1 and 2: non-concentrated CM. Lanes 3 and 4: concentrated CM, without EDTA. Lanes 5 and 6:concentrated CM, with EDTA. Lanes 7 and 8: CM samples concentrated without EDTA, but incubated with 10mM EDTA during the development of the proteinase bands on the gel.
Role of extracellular metalloproteinases in regulation of proliferation
The possibility that the secreted MPs were part of a growth-stimulatory loop seemed
reasonable since MPs are known to be involved in autocrine loops regulating cell
growth and differentiation, among other processes (Fowlkes and Winkler 2002). To
study a possible correlation between cell proliferation and MP activity, an
experimental approach was developed involving a specific MP inhibitor, DL-
thiorphan, which had been shown to completely inhibit the MP activity. DL-thiorphan
is a small organic molecule which may penetrate the cell membrane if added directly
to cultures, and thus heavily complicate the interpretation of the data. Instead, CM was
incubated with DL-thiorphan for 24 h and excess inhibitor was removed prior to
supplementation of the CM to new cultures. As expected, CM without inhibitor
treatment had a clear stimulatory effect on cell proliferation. On the contrary, no
growth-promoting effect at all could be observed with inhibitor-treated CM (Fig. 13).
A control culture with inhibitor-treated fresh medium showed that inhibitor treatment
per se had no negative effect on cell proliferation. Thus, these data strongly indicate
that the MPs play a major role in the regulation of proliferation in serum-free cultures.
Indeed, this is the first report on a correlation between MP activity and the growth-
promoting effect of CM on T. ni cells. It is not clear whether all forms of the MP or
29
just the main form at 48 kDa are involved in the regulation, and this remains to be
elucidated.
0
2
4
6
0 40 80 120 160
Via
ble
cell
dens
ity (
106 c
ells
ml-1
)
Culture time (h)
Figure 13. Influence on cell growth by addition of 20 % CM pre-treated by DL-thiorphan () and 20 % CMwithout inhibitor treatment (). Cultures supplemented with fresh medium pre-treated by DL-thiorphan () andfresh medium alone (Ο) were used as controls.
Metalloproteinase activity also correlates to the inoculum cell concentration. Samples
were taken at 21 and 48 h from cultures inoculated between 0.3 x 106 and 3 x 106 cells
ml-1 in fresh medium. The results showed that, although faint, proteinase activities
detected at 21 h increased with increasing inoculum cell concentration in all cultures
except for the two lowest inoculum cell concentrations, where no MP activity could be
detected (Fig. 14a). At 48 h, the MP activity had increased in all cultures and a weak
proteinase band could also be detected in the culture inoculated at the lowest cell
concentration (Fig. 14b). The appeared band at 48 h had a slightly lower molecular
mass than the band observed at 21 h, although both bands fell within a molecular mass
range of 45 to 55 kDa. As previously discussed, the MP exists in several different
molecular mass forms due to autoproteolysis; thus, these bands correspond to different
molecular mass forms of the same enzyme. The significance of the different forms is
not yet known. As noted before, there was a relationship between inoculum cell
30
density and lagphase length, the theory being that cultures inoculated with higher cell
concentrations start to produce more MP than the other cultures, leading to a faster
accumulation up to the critical MP concentration necessary for initiation of
proliferation. Hence, this result further strengthen the role of MP as the growth-
promoting factor in CM.
Figure 14. Proteolytic activity in cultures inoculated at different cell concentrations as visualized by caseinzymography. Samples were taken after (a) 21 h and (b) 48 h of culture. Inoculation densities: 0.3 x 106 cells ml-1
(lane 1), 0.5 x 106 cells ml-1 (lane 2), 0.7 x 106 cells ml-1 (lane 3), 1.0 x 106 cells ml-1 (lane 4), 2.0 x 106 cells ml-1
(lane 5), and 3.0 x 106 cells ml-1 (lane 6).
Thus, the question arises how this regulation mechanism functions. The activities of
growth factors and cytokines are commonly restricted by association to extracellular
matrix (ECM) proteins or to other binding proteins, and the availability of these factors
for ligand-binding is regulated by proteolytic processing of the various protein
complexes. Matrix MPs (MMP) belong to the metzincin superfamily and are well
known as the major player in regulation of ECM turnover (Taipale and Keski-Oja
1997; Fowlkes and Winkler 2002). Furthermore, MMPs, together with other members
of this family, are involved in release of growth factors and cytokines from their
association with other proteins (Fowlkes et al. 1999; Killar et al. 1999; Fowlkes and
Winkler 2002). They are for example involved in liberation of insulin-like growth
factors (IGF) from their binding proteins (IGFBP). This is very interesting as the
presence of IGFBP in CM from T. ni is already established (Andersen et al. 2000;
Doverskog et al. 1999). The functions of IGF is to regulate cell proliferation and
prevent apoptosis through autocrine and paracrine mechanism, and to mediate the
31
effects of a growth hormone. IGF action is highly regulated through binding to specific
IGFBP and thus, very little IGF exist in “free” form (Jones and Clemmons 1995). The
degradation of IGFBP by MPs is thought be an important part in the regulation of IGF
activity. Further, MPs have been suggested to regulate IGF-mediated proliferation in
cell cultures and tissue growth in vitro (Fowlkes et al. 1999; Sadowski et al. 2003).
Thus, it is tempting to speculate that the binding protein secreted by the T. ni cells
functions as the target protein for the MP and that degradation of the IGFBP releases a
sequestered ligand, which binds to cell surface receptors and exerts a mitogenic effect
(Fig. 15). Moreover, MP activity in general is highly regulated through feed-back
mechanisms via inhibitors (Woessner and Nagase 2000), and the activity of the MP
found in T. ni cultures may be controlled by the secreted proteinase inhibitor
(identified in the growth-inhibitory fraction).
Figure 15. Hypothesis of the stimulatory effect on cell growth exerted by the secreted metalloproteinase (MP),the theory being that MPs degrade IGFBPs and release free IGFs (or other growth factors), which then bind tocell surface receptors and stimulate proliferation. This loop is regulated by a proteinase inhibitor, which preventsthe degradation of the IGFBP by the MP.
An alternative explanation to the growth-promoting effect of the MP could be that
proteolysis of extracellular proteins releases amino acids which enhances proliferation.
This appears less likely to be the case since the growth-promoting effect of CM is
rather prominent in the beginning of culture when the medium is still rich in nutrients.
Another option might be that the yeast extract present in Express Five SFM contains
proteins/peptides which could be cleaved by the MP to liberate a peptide with growth
32
stimulatory properties. This seems unlikely as well, as CM filtrated through a 10 kDa
cut-off filter did not have any effect on cell proliferation.
However, this is all speculations and to fully understand how this regulatory system
functions further work has to be done. Although purification of the MP from CM
would have been a first step in this strategy, the development of a purification process
for the MP was proven to be extremely difficult.
Recombinant protein production
Baculovirus expression vector system
The baculovirus expression vector system (BEVS) as a gene delivery tool for insect
cells was first introduced by Smith and his coworkers in 1983 and have since then
become one of the most versatile and powerful eukaryotic vector systems for
recombinant protein production (Luckow and Summers 1988; Kost and Condreay
1999). Some of the advantages are that baculovirus are non-hazardous to vertebrates
and that the expression levels can be very high; concentrations up to 500 mg L-1 have
been reported (Beljelarskaya 2002). Further, the large size of the viral genome enables
insert of rather big genes and consequently expression of large and complex proteins.
BEVS is based on the introduction of a foreign gene into the viral genome and
virtually any kind of gene can be inserted via homologous recombination. This
characteristic proposes a great flexibility of the BEVS (Luckow and Summers 1988;
Kitts and Possee 1993). Since BEVS is used in eukaryotic cells, many of the post-
translational modifications that mammalian cells do, such as phosphorylation and
glycosylation, are performed (with some exceptions). Further processing of the protein
product such as cleavage of signal peptides, disulfide-bond formations and correct
folding is also similar to mammalian cells, and this in conjunction with a conserved
protein targeting process, heavily contributes to biologically active and correctly
folded proteins (Luckow and Summers 1988)
33
Productivity – Conditioned medium factors and cell cycle dynamics
In spite of all the advantages associated with BEVS some significant drawbacks are
linked to this system. One of the major problems is that the specific productivity
decreases as the cell density for infection increases, the so called cell density effect.
This cell density dependent drop in productivity has been observed for both Sf9
(Caron et al. 1990; Lindsay and Betenbaugh 1992; Taticek and Shuler 1997;
Doverskog et al. 2000) and T. ni insect cells (Dee et al. 1997; Donaldson and Shuler
1998a; Chico and Jäger 2000; Taticek et al. 2001). For T. ni cells, cell densities with
maximum productivity has been suggested not to exceed 1 x 106 cells ml-1 which is
rather low in comparison to the final cell concentrations that can be reached.
The probable causes behind the cell density dependent decrease in productivity have
been extensively studied by several research groups; however, there is still no clear
explanation, although accumulation of metabolic by-products or nutrient depletion
have been considered as potential causes (Yang et al. 1996; Chico and Jäger 2000). As
mentioned before, Yang et al. (1996) noticed that glutamine and asparagine were
among the first nutrients to be consumed in a T. ni culture; however, Chico and Jäger
(2000) could show that recombinant protein production continued for more than 60 h
after glutamine and asparagine depletion. Further, the impact of high ammonia
accumulation on protein productivity was suggested to be negative by some research
groups (Yang et al. 1996; Donaldson et al. 1999), whereas no direct effect could be
observed by other groups (Chico and Jäger 2000). Thus, research within this field
shows some inconsistency and so far no limiting nutrients or toxic by-products have
been identified as the cause behind the cell density effect. Different strategies have
been addressed to improve protein production in T. ni insect cell cultures, such as
process optimization, medium exchange prior to infection, specific nutrient
supplementations and different feeding strategies (Chico and Jäger 2000; Wang and
Doong 2000; Ikonomou et al. 2004).
34
One of the objectives of this work was to study the productivity of T. ni insect cells
and parameters influencing productivity during serum-free conditions (paper II).
Samples were taken from a culture at different time-points and infected with
recombinant baculovirus containing the gene for the ligand-binding domain of the
glucocorticod receptor (GR-LBD). The results showed that for cells inoculated in 100
% fresh medium there was an increase in specific productivity (measured as (µg
protein (108 cells, 48 h)-1)) during the first 7 hours of culture, after which a rapid
decline in specific productivity could be observed (Fig. 16). Thus, the specific
productivity peak occurred after only 7 hours when the cell density was still very low.
Nutrient depletion seemed very unlikely at this time-point and metabolic analysis
confirmed that there were still plenty of nutrients left in the culture (Figs. 4 and 5). The
low amount of by-products accumulated this early in culture is not known to have a
negative effect on recombinant protein production. Thus, these results exclude nutrient
limitation and accumulation of toxic by-products as possible causes for the cell density
effect observed here.
0
400
800
1200
0
2
4
6
0 40 80 120 160
Spec
ific
prod
uctiv
ity
(µg
prot
ein
(108 c
ells,
48
h)-1
)
Viable cell desnity (10 6 cells m
l -1)
Culture time (h)
Figure 16. The effect of culture time and cell density (Ο) on specific protein productivity () in a T. ni culture in100 % fresh Express Five SFM. The dramatic drop in productivity can be seen after 7 hours of culture and a celldensity of 2 x 105 cells ml-1.
35
Productivity in serum-free insect cell cultures has also been proposed to be dependent
on autoregulatory events. A link between proliferation and autocrine factors has
previously been established for Sf9 cells (Doverskog et al. 2000). Recent results
showed that CM from Sf9 cultures had a negative effect on the protein production,
although CM did not affect protein production per se, but rather through its effect on
cell physiology (personal communication, Karin Calles). On the contrary, stimulatory
effects of CM from T. ni cultures on protein productivity have been reported for T. ni
cells (Ikonomou et al. 2004). They showed that 25 % CM had a beneficial effect on
protein production, whereas higher amount of CM had no effect. However, the
infection studies were performed at one single time-point and at a rather high cell
density (4.2 x 106 cells ml-1), in contrast to our study in which infection studies took
place at a broad range of cell densities.
In cultures supplemented with 20 % CM, the specific productivity was maintained
much longer than in cultures with 100 % fresh medium (Fig. 17a). The drop in
productivity did not occur until a cell concentration of 1 x 106 cell ml-1, in contrast to
the fresh-medium culture where the productivity dropped in relation to increasing cell
densities (Fig. 17b). A clear positive effect on specific productivity by CM was
observed between 20 and 72 hours of culture, with a 30 % increase in per cell
productivity (Fig. 17). The product concentration was also around 30 % higher and the
maximum product concentration was obtained on day two, rather than on day three as
for the reference culture (Fig. 17c).
36
0
400
800
1200
0 20 40 60 80 100
Spec
ific
prod
uctiv
ity
(µg
prot
ein
(108 c
ells,
48
h)-1
)
Culture time (h)
a
0
400
800
1200
0.2 0.35 0.5 1 2 3 4
Spec
ific
prod
uctiv
ity
(µg
prot
ein
(108 c
ells,
48
h)-1
)
Cell density at the time of infection (106 cells ml-1)
b
0
2
4
6
8
10
12
14
16
0 40 80
Prod
uct c
once
ntra
tion
(mg
L-1)
Culture time (h)
cFigure 17. Protein productivity in T. ni culturessupplemented with 20 % CM as compared to a referenceculture in 100 % fresh medium. (a) Specific productivity;20 % CM () and 100 % fresh medium (Ο). (b) Effect ofthe cell density at the time of infection on the specificproductivity. Dotted columns represent the referenceculture in 100 % fresh medium and the filled columnsrepresent the culture supplemented with 20 % CM. Thedata are based on the values in (a). (c) Productconcentrations; 20 % CM () and 100 % fresh medium(Ο).
Important to notice is that the beneficial effect of CM on productivity may vary
depending on different compositions of CM and on the state of the cell culture, as
previously discussed for cell growth. Nevertheless, these data clearly show that CM
contains factors with a stimulatory effect on recombinant protein production. The
positive effect of CM on specific productivity observed after 20 hours coincided with
the time a stimulatory effect on cell growth was first observed. Thus, it appears as the
productivity may be determined by the proliferation potential of the culture. As the
stimulatory effect on cell proliferation already has been ascribed to the secreted MP, it
is appealing to consider that the same MP indirectly would affect the productivity.
Again, these are merely speculations and the effect of CM factors on protein
production remains to be investigated.
37
Cell diameter (or cell volume) has also been shown to have a significant effect on
productivity, and hence, has been proposed as a powerful tool for predicting
recombinant protein production (Palomares et al. 2001). It has been postulated that the
cell size before infection correlates with the potential of a cell line to produce
recombinant proteins and that larger cells are able to express more proteins (Chai et al.
1996). However, this statement disagrees with our results which showed that the
smaller sized CM-cells produce more recombinant proteins per cell for a longer period
of time than normal cells.
Finally, the cell cycle phase at the time of infection is another parameter that might
influence the productivity. Productivity may be affected by the distribution of cells in
different cell cycle phases. For example, Lynn and Hink (1978b) showed that
synchronized cultures, which were infected during the middle and late S phase, had
higher percentage of infected cells than cultures infected in the G2 phase. Similar
observations have been made with the Sf9 cells (Kioukia et al. 1995). Even though our
data did not provide a very clear correlation between distinct cell cycle phases and
production capacity, a higher specific productivity could be observed for cell
populations with higher fractions of cells in the S phase. Consequently, this is in
agreement with previous studies (Lynn and Hink 1978b).
38
Conclusions and future perspectives
In addition to the engineering aspects of the bioprocess, knowledge of insect cell
metabolism and physiology is essential for improvements of the recombinant protein
production process. Insect cell lines may differ a great deal in metabolism, physiology
and nutritional requirements.
Results presented in this study show that a variety of parameters control cell
proliferation and productivity. One important mechanism for cell proliferation of T. ni
insect cells in serum-free cultures has been ascribed to an autocrine regulation system;
that is, T. ni cells produce autocrine factors which affect their proliferation. Addition
of CM to new cultures presents a clear growth-stimulatory effect on cell growth. The
coordinated effects of CM on cell cycle distribution, cell size and culture lagphase
confirm the stimulatory properties of CM and suggest a correlation between CM
factors and changes in the cell cycle distribution. A secreted MP has been proposed to
play a significant role in the growth stimulatory autocrine loop. One might speculate
that the secreted IGFBP and the proteinases inhibitor are involved in this regulation
system. However, since many other proteins are secreted as well the situation may be
even more complex. Autocrine loops involving MPs tend be quite complex, involving
regulatory elements such as autoproteolytic degradation and different inhibitors.
T. ni also produce some factors with a beneficial effect on protein production. Cell
cultures supplied with CM exhibited a higher specific productivity in relation to time
and cell density, as compared to cultures without CM. Although no proteins have been
identified as the factor responsible for this positive effect, it is tempting to speculate
that the secreted MP would be involved in some way. This is particularly interesting
since the positive effect on productivity was shown to coincide with a positive effect
on cell growth. Although CM factors play an important role in cell proliferation and
productivity, these systems are very complex and depend on other factors as well, such
as cell cycle dynamics, nutrient availability and suitable environmental conditions.
39
However, these data propose an alternative explanation for the observed cell density
dependent decrease in productivity; nutrient limitation and accumulation of toxic by-
products have been eliminated as potential causes in this study.
A full understanding of the physiological roles of the secreted MP requires cloning and
sequencing of the protein. When the mechanisms behind the autocrine system are fully
explored it may be possible to control the cells’ production of the relevant factors and
hence, control the proliferation in a way that is adequate for optimal baculovirus
expression.
40
Acknowledgements
I would like to thank everyone that in some way has contributed to this work, and I wouldespecially like to thank the following:
Professor Lena Häggström, for being an excellent supervisor and for making all this possibleby accepting me as a PhD student. Thank you for sharing your knowledge, good advice,always being open to discuss results and ideas, and for being enthusiastic and inspiring nomatter what.
The ‘animal cell girls’ for your encouragement, help and support during these years, and allthe laughter and great times we have had both inside and outside of the lab. Your friendshipand support have meant a lot. Eva, for your help, guidance and good discussions. Erika, forbeing an excellent conference partner and for all the fun we had in Granada and Cancun, forgreat talks and for taking care of my cells. I owe you! Karin, for a great collaboration and forintroducing me to the surroundings of ‘Flempan’, even if it meant getting lost, and for greatfun in Granada. Ingrid, for your friendship and being a great room mate, for daily chats andbeing there to listen, a great climbing partner and all the fun we have had on our trips.
All former and present colleagues at Bioprocess Technology for creating a friendlyatmosphere and for always being helpful. Special thanks for all the laughter and great timesduring these years, both at the lab and at after work activities. I have many good memories!Thank you Ela for the glucose analysis and Thep for all the help with the flow cytometer.
Thanks to the senior researchers at our department for sharing your knowledge and beingopen for discussions. Especially associate professor Gen Larsson for reading my thesis.
AstraZeneca, Global Protein Science and Supply, for supporting this project.
Karo Bio AB, and especially the Structural Biology group, for general cooperation and formaking me feel welcome and part of the group during the weeks I spent there.
My friends, for being supportive and taking my mind away from work from time to time.
My family, for your support and faith in me, and for always being there.
Finally Ulf, my partner through everything, for all the love, support and faith in me duringthese years .You believed in me when I needed it the most. You mean the world to me!
41
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