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.

2

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

3

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.

4

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.

5

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

6

(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

7

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

8

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.

9

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.

10

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.

11

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.

12

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.

13

0

5

10

15

20

0

0.5

1

1.5

2

0 40 80 120

Asp

arag

ine

and

glut

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”).

14

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

15

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

References

Agathos S (1996). Insect cell bioreactors. Cytotechnology 20: 173-189.

Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson, JD (1994). Molecular biology of the cell.New York, Garland Publishing, Inc.

Andersen A, Hansen P, Schäffer L and Kristensen C (2000). A new secreted insect protein belongingto the immunoglobulin superfamily binds insulin and related peptides and inhibits their activities. J.Biol. Chem. 275: 16948-16953.

Auzoux-Bordenave S, Hatt P and Porcheron P (2002). Anti-proliferative effect of 20-hydroxyecdysone in a lepidopteran cell line. Insect Biochem. Molec. 32: 217-223.

Baines D (1996). New approaches to insect tissue culture. Cytotechnology 20: 13-22.

Bédard C, Kamen A, Tom R and Massie, B (1994). Maximization of recombinant protein yield in theinsect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures.Cytotechnology 15: 192-238.

Bédard C, Perret S and Kamen A (1997). Fed-batch culture of Sf-9 cells supports 3x107 cells per mland improves baculovirus-expressed recombinant protein yields. Biotechnol. Lett. 19: 629-632.

Bédard C, Tom R and Kamen A (1993). Growth, nutrient consumption, and end-product accumulationin Sf-9 and BTI-EAA insect cell cultures: insight into growth limitation and metabolism. Biotechnol.Prog. 9: 615-624.

Beljelarskaya S (2002). A baculovirus expression system for insect cells. Mol. Biol. 36: 371-385.

Bhatia R, Jesionowski G, Ferrance J and Ataai M (1997). Insect cell physiology. Cytotechnology 24:1-9.

Brogiolo W, Stocker H, Ikeya T, Rintelen F, Fernandez R and Hafen, E (2001). An evolutionaryconserved function of the Drosophila insulin receptor and insulin-like peptids in growth control. Curr.Biol. 11: 213-221.

Caron A, Aramchambault J and Massie B (1990). High-level recombinant protein production inbioreactors using the baculovirus-insect cell expression system. Biotechnol. Bioeng. 36: 1133-1140.

Cassier P, Serrant P, Garcia R, Coudouel N, Andre M, Guillaumin D, Porcheron P and Oberlander H(1991). Morphological and cytochemical studies of the effects of ecdysteroids in a lepidopteran cellline (IAL-PID2). Cell Tissue Res. 265: 361-369.

Chai H, Al-Rubeai M, Chua K, Oh S and Yap M (1996). Insect cell line dependent gene expression ofrecombinant human tumor necrosis factor-β. Enzyme Microbiol. Technol. 18: 126-132.

Chan L, Young P, Bletchly C and Reid S (2002). Production of the baculovirus-expressed denguevirus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch culturesand the addition of the insect moulting hormone, 20-Hydroxyecdysone. J. Virol. Methods 105: 87-98.

42

Chico E and Jäger V (2000). Perfusion culture of baculovirus-infected BTI-Tn-5B1-4 insect cells: amethod to restore cell-specific β-trace glycoprotein productivity at high cell density. Biotechnol.Bioeng. 70: 574-586.

Coleman T and Dunphy W (1994). Cdc2 regulatory factors. Curr. Opin. Cell Biol. 6: 877-882.

Cruz P, Martins P, Alves P, Peixoto C, Santos H, Moreira J and Carrondo M (1999). Proteolyticactivity in infected and noninfected insect cells: degradation of HIV-1 Pr55gag particles. Biotechnol.Bioeng. 65: 133-143.

Davis K and Shearn A (1977). In vitro growth of imaginal disks from Drosophila melanogaster.Science 196: 438-440.

Davis T, Wickham T, McKenna K, Granados R, Shuler M and Wood H (1993). Comparativerecombinant protein production of eight insect cell lines. In Vitro Cell Dev. Biol. 29: 399-390.

Dee K, Shuler M and Wood H (1997). Inducing single-cell suspension of BTI-Tn5B1-4 insect cells: I.The use of sulfated polyanions to prevent cell aggregation and enhance recombinant proteinproduction. Biotechnol. Bioeng . 54: 191-205.

DiFalco M, Bakopanos E, Patricelli M, Chan G and Congote L (1997). The influence of various insectcell lines, p10 and polyhedrin promotors in the production of secreted insulin-like growth factor-interleukin-3 chimeras in the baculovirus expression system. J. Biotechnol. 56: 49-56.

Donaldson M and Shuler M (1998a). Low-cost serum-free medium for the BTI-Tn5B1-4 insect cellline. Biotechnol. Prog. 14: 573-579.

Donaldson M and Shuler M (1998b). Effects of long-term passaging of BTI-Tn5B1-4 insect cells ongrowth and recombinant protein production. Biotechnol. Prog. 14: 543-547.

Donaldson M, Wood H, Kulakosky P and Shuler M (1999). Glycosylation of a recombinant protein inthe Tn5B1-4 insect cell line: Influence of ammonia, time of harvest, temperature, and dissolvedoxygen. Biotechnol. Bioeng . 63: 255-262.

Doverskog M, Bertram E, Ljunggren J, Öhman L, Sennerstam R and Häggström L (2000). Cell cycleprogression in serum-free cultures of Sf9 insect cells: modulation by conditioned medium factors andimplications for proliferation and productivity. Biotechnol. Prog. 16: 837-846.

Doverskog M, Han L and Häggström L (1998). Cystine/cysteine metabolism in cultured Sf9 cells:influence of cell physiology on biosynthesis, amino acid uptake and growth. Cytotechnology 26: 91-102.

Doverskog M, Tally M and Häggström L (1999). Constitutive secretion of an endogenous insulin-likepeptide binding protein with high affinity for insulin in Spodoptera frugiperda (Sf9) cell cultures.Biochem. Biophys. Res. Co. 265: 674-679.

Drews M, Paalme T and Vilu R (1995). The growth and nutrient utilization of the insect cell lineSpodoptera frugiperda Sf9 in batch and continuous culture. J. Biotechnol. 1995: 187-198.

English L, Magelky B and Marks E (1984). 20-hydroxyecdysone-induced changes in the cell volumeof lepidopteran cells associated with population dynamics. In Vitro 20: 71-78.

Ferkovich S and Oberlander H (1991). Growth factors in invertebrate in vitro culture. In Vitro CellDev. Biol. 27: 483-486.

43

Fernandez J and Hoeffler J (Eds.) (1999). Gene expression systems: using nature for the art ofexpression. Academic Press,

Ferrance J, Goel A and Ataai M (1993). Utilization of glucose and amino acids in insect cell cultures:quantifying the metabolic flows within the primary pathways and medium development. Biotechnol.Bioeng. 42: 697-707.

Fertig G, Klöppinger M and Miltenburger H (1990). Cell cycle kinetics of insect cell culturescompared to mammalian cell cultures. Exp. Cell Res. 189: 208-212.

Fowlkes J, Serra D, Nagase H and Thrailkill K (1999). MMPs are IGFBP-degrading proteinases:implications for cell proliferation and tissue growth. Ann. NY Acad. Sci. 878: 696-699.

Fowlkes J and Winkler M (2002). Exploring the interface between metalloproteinase activity andgrowth factor and cytokine bioavailability. Cytokine Growth. F. R. 13: 277-287.

Fujimura S, Oshikawa K, Terada S and Kimoto E (2000). Primary structure and autoproteolysis ofbrevilysin H6 from the venom of Gloydius halys brevicaudus. J. Biochem. 128: 167-173.

Fullbright G, Lacy E and Bullesbach E (1997). The prothoracicotropic hormone bombyxin hasspecific receptors on insect ovarian cells. Eur. J. Biochem. 245: 774-780.

Fussenegger M and Bailey J (1998). Molecular regulation of cell-cycle progression and apoptosis inmammalian cells: Implications for biotechnology. Biotechnol. Prog. 14: 807-833.

Gade G, Hoffmann K and Spring J (1997). Hormonal regulation in insects: facts, gaps and futuredirections. Physiol. Rev. 77: 963-1032.

Grace T (1962). Establishment of four strains of cells from insect tissues grown in vitro. Nature 195:788-789.

Granados R, Guoxun L, Derksen A and McKenna K (1994). A new insect cell line from Trichoplusiani (BTI-Tn-5B1-4) susceptible to Trichoplusia ni single enveloped nuclear polyhedrosis virus. J.Invertebr. Pathol. 64: 260-266.

Guttieri M, Bookwalter C and Schmaljohn C (2000). Expression of a human, neutralizing monoclonalantibody specific to Puumala virus G2-protein in stably-transformed insect cells. J. Immunol. Methods.246: 97-108.

Hatt P, Liebon C, Morinière M, Oberlander H and Porcheron P (1997). Activity of insulin growthfactors and shrimp neurosecretory organ extracts on a Lepidopteran cell line. Arch. Insect Biochem.34: 313-328.

Hayakawa Y and Ohnishi A (1998). Cell growth activity of growth-blocking peptide. Biochem. Bioph.Res. Co. 250: 194-199.

Hensler W, Singh V and Agathos S (1994). Sf9 insect cell growth and β-galactosidase production inserum and serum-free media. Ann. NY Acad. Sci. 745: 149-168.

Hink W (1970). Established insect cell line from the cabbage looper, Trichoplusia ni. Nature 226:466-467.

Hink W, Thomsen D, Davidson D, Meyer A and Castellino F (1991). Expression of three recombinantproteins using baculovirus vectors in 23 insect cell lines. Biotechnol. Prog. 7: 9-14.

44

Hu W and Aunins J (1997). Large-scale mammalian cell culture. Curr. Opin. Biotechnol. 8: 148-153.

Ikonomou L, Bastin G, Schneider Y and Agathos S (2001). Design of an efficient medium for insectcell growth and recombinant protein production. In Vitro Cell Dev. Biol. - Animal 37: 549-559.

Ikonomou L, Bastin G, Schneider Y and Agathos S (2004). Effect of partial medium replacement on

cell growth and protein production for the High-FiveTM insect cell line. Cytotechnology 44: 67-76.

Ikonomou L, Peeters-Joris C, Schneider Y and Agathos S (2002). Supernatant proteolytic activities ofHigh-Five insect cells grown in serum-free culture. Biotechnol. Lett. 24: 965-969.

Ikonomou L, Schneider Y and Agathos S (2003). Insect cell culture for industrial production ofrecombinant proteins. Appl. Microbiol. Biotechnol. 62: 1-20.

Iwami M, Tanaka A, Hano N and Sakurai S (1996a). Bombyxin gene expression in tissues other thanbrain detected by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization.Experientia 52: 882-887.

Iwami M, Furuya I and Kataoka H (1996b). Bombyxin-related peptides: cDNA structure andexpression in the brain of the hornworm Agrius convoluvuli. Insect Biochem. Mol. Biol. 1(25-32).

Jin Z, Melaragno M, Liao D, Yan C, Haendeler J, Suh Y, Lambeth J and Berk B (2000). CyclophilinA is a secreted growth factor induced by oxidative stress. Circ. Res. 87: 789-796.

Jones J and Clemmons D (1995). Insulin-like growth factors and their binding proteins: biologicalactions. Endocr. Rev. 16: 3-34.

Jäger V (1996). Perfusion bioreactors for the production of recombinant proteins in insect cells.Cytotechnology 20: 191-198.

Kang D, Liu G, Gunne H and Steiner H (1996). PCR differential display of immune gene expressionin Trichoplusia ni. Insect Biochem. Mol. Biol. 26: 177-184.

Kawamura K, Shibata T, Saget O, Peel D and Bryant P (1999). A new family of growth factorsproduced by the fat body and active on Drosophila imaginal disc cells. Development 126: 211-219.

Killar L, White J, Black R and Peschon J (1999). Adamalysins. A family of metzincins includingTNF-α converting enzyme (TACE). Ann. NY Acad. Sci. 878: 442-452.

Kioukia N, Nienow A, Emery A and Al-Rubeai M (1995). Physiological and environmental factorsaffecting the growth of insect cells and infection with baculovirus. J. Biotechnol. 38: 243-251.

Kitts P and Possee R (1993). A method for producing recombinant baculovirus expression vectors athigh frequency. BioTechniques 14: 810-817.

Kleiner D and Stetler-Stevenson W (1994). Quantitative zymography: detection of picogram quantitiesof gelatinases. Anal. Biochem. 218: 325-329.

Komano H, Homma K and Natori S (1991). Involvement of sapecin in embryonic cell proliferation ofSarcophaga peregrina (flesh fly). FEBS Lett. 289: 167-170.

Kost T and Condreay J (1999). Recombinant baculoviruses as expression vectors for insect andmammalian cells. Curr. Opin. Biotechnol. 10: 428-433.

45

Koths K (1995). Recombinant proteins for medical use: the attractions and challenges. Curr. Opin.Biotechnol. 6: 681-687.

Kramer K (1985). Vertebrate hormones in insects. Oxford, Oxford Pergamon.

Krieger M, Jahan N, Riehle M, Cao C and Brown M (2004). Molecular characterization of insulin-likepeptide genes and their expression in the African malaria mosquito, Anopheles gambiae. Insect Mol.Biol. 13: 303-315.

Lauffenburger D and Cozens C (1989). Regulation of mammalian cell growth by autocrine growthfactors: analysis of consequences for inoculum cell density effects. Biotechnol. Bioeng. 33: 1365-1378.

Lindsay D and Betenbaugh M (1992). Quantification of cell culture factors affecting recombinantprotein yields in baculovirus-infected cells. Biotechnol. Bioeng. 39: 614-618.

Luckow V and Summers M (1988). Trends in the development of baculovirus expression vectors.Bio/Technology 6: 47-55.

Lynn D (1996). Development and characterization of insect cell lines. Cytotechnology 20: 3-11.

Lynn D and Hink W (1978a). Cell cycle analysis and synchronization of the Tn-368 insect cell line. Invitro 14: 236-238

Lynn D and Hink W (1978b). Infection of synchronized TN-368 cell cultures with alfalfa loopernuclear polyhedrosis virus. J. Inv. Pathol . 32: 1-5.

Maiorella B, Inlow D, Shauger A and Harano D (1988). Large-scale insect cell-culture forrecombinant protein production. Bio/Technology 6: 1406-1410.

Morgan D (1995). Priciples of CDK regulation. Nature 374: 131-134.

Mosna G (1981). Insulin can completely replace serum in Drosophila melanogaster cell cultures invitro. Experientia 37: 466-467.

Nagase H (1997). Activation mechanisms of matrix metalloproteinases. Biol. Chem. 378: 151-160.

Nguyen B, Jarnagin K, Williams S, Chan H and Barnett J (1993). Fed-batch culture of insect cells: amethod to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirusexpression system. J. Biotechnol. 31: 205-217.

Nijhout H (1994). Insect hormones. Princeton, New Jersey, Princeton University Press.

Nijhout H and Grunert L (2002). Bombyxin is a growth factor for wing imaginal disks in Lepidoptera.PNAS 99: 15446-15450.

Nishino H and Mitsuhashi J (1995). Effects of some mammalian growth promoting substances oninsect cell cultures. In Vitro Cell Dev. Biol. - Animal 32: 822-823.

Palomares L, Pedroza J and Ramírez O (2001). Cell size as a tool to predict the production ofrecombinant protein by the insect-cell baculovirus expression system. Biotechnol. Lett. 23: 359-364.

Porcheron P, Morinière M, Coudouel N and Oberlander H (1991). Ecdysteroid-stimulated synthesisand secretion of N-acetyl-D-glucosamine rich glycopeptide in a lepidopteran cell line from imaginaldiscs. Arch. Insect Biochem. Physiol. 16: 257-271.

46

Ramírez O and Mutharasan R (1990). Cell cycle and growth phase-dependent variations in sizedistributions, antibody productivity, and oxygen demand in hybridoma cultures. Biotechnol. Bioeng.36: 839-848.

Rhiel M, Mitchell-Logean C and Murhammer D (1997). Comparison of Trichoplusia ni BTI-Tn-5B14(High Five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures.Biotechnol.. Bioeng.. 55: 909-920.

Runstadler P (1992). The importance of cell physiology to the performance of animal cell bioreactors.Ann. Acad. Sci. NY 665: 380-390.

Sadowski T, Dietrich S, Koschinsky F and Sedlacek R (2003). Matrix metalloproteinase 19 regulatesinsulin-like growth factor-mediated proliferation, migration, and adhesion in human keratinocytesthrough proteolysis of insulin-like growth factor binding protein-3. Mol. Biol. Cell. 14: 4569-4580.

Sárvári M, Csikós G, Sass M, Gál P, Schumaker V and Závodszky P (1990). Ecdysteroids increase theyield of recombinant proteins produced in baculovirus insect cell expression system. Biochem.Biophys. Res. Co. 167: 1154-1161.

Sasagawa H, Nakahara Y and Kiuchi M (2001). An ENF peptide, Bombyx mori paralytic peptide,induces cell proliferation and morphological changes in Bombyx cell lines. In Vitro Cell Dev. - Animal36: 638-640.

Schlaeger E (1996). Medium design for insect cell culture. Cytotechnology 20: 57-70.

Schlaeger E, Goggetta M, Vonach J and Christensen K (1993). Sf-1 a low cost culture medium for theproduction of recombinant proteins in baculovirus infected insect cells. Biotechnol. Tech. 7: 183-188.

Seamon K (1998). Specifications for biotechnology-derived protein drugs. Curr. Opin. Biotechnol. 9:319-325.

Smith G, Summers M and Fraser M (1983). Production of human beta interferon in insect cellsinfected with a baculovirus expression vector. Mol. Cell Biol. 3: 2156-2165.

Stockdale H and Gardiner G (1976). Utilization of some sugars by a line of Trichoplusia Ni cells.Invertebrate Tissue Culture. Kurstak E and Maramorosh K (eds.). Academic Press, London.

Taipale J and Keski-Oja J (1997). Growth factors in the extracellular matrix. FASEB J. 11: 51-59.

Taticek R, Choi C, Phan S, Palomares L and Shuler M (2001). Comparison of growth and recombinantprotein expression in two different insect cell lines in attached and suspension culture. Biotechnol.Prog. 17: 676-684.

Taticek R and Shuler M (1997). Effect of elevated oxygen and glutamine levels on foreign proteinproduction at high cell denities using the insect cell-baculovirus system. Biotechnol. Bioeng. 54: 142-152.

Trexler M, Bányai L and Patthy L (2001). A human protein containing multiple types of protease-inhibitory modules. PNAS 98: 3705-3709.

Vaughn J, Goodwin R, Tompkins G and McCawley P (1977). The establishment of two cell lines fromthe insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13(213-217).

47

Wang M and Doong S (2000). A pH-based fed-batch process for the production of a chimericrecombinant infectious bursal disease virus (IBDV) structural protein (rVP2H) in insect cells. ProcessBiochem. 35: 877-884.

Weiss S, Smith G, Kalter S and Vaughn J (1981). Improved method for the production of insect cellcultures in large volumes. In Vitro 17: 495-502.

Wilkie G, Stockdale H and Pirt S (1980) Chemically-defined media for production of insect cells andviruses in vitro. Dev. Biol. Stand. 46: 29-37.

Woessner J and Nagase H (2000). Matrix metalloproteinases and TIMPs. New York, Oxford UnivPress.

Wu J, King G, Daugulis A, Faulkner P, Bone D and Goosen M (1989). Engineering aspects of insectcell suspension culture: a review. Appl. Microbiol. Biotechnol. 32: 249-255.

Wu J and Lee K (1998). Growth promotion by yeastolate and related components on insect cells.Biotechnol. Tech. 12: 67-70.

Yang J, Gecik P, Collins A, Czarnecki S, Hsu H, Lasdun A, Sundaram R, Muthukumar G andSilberklang M (1996). Rational scale-up of a baculovirus-insect cell batch process based on mediumnutritional depth. Biotechnol. Bioeng. 52: 696-706.

Öhman L, Alarcon M, Ljunggren J, Ramqvist AK and Häggström L (1996). Glutamine is not anessential amino acid for Sf-9 insect cells. Biotechnol. Lett. 18: 765-770.