Ebook Update on production of recombinant therapeutic protein – Transient gene expression: Part 1

Smithers Rapra Technology Ltd, 2013

Over the past decade, the transient gene expression (TGE) technology platform
has been actively pursued to produce a wide range of therapeutic proteins,
monoclonal antibodies, and vaccines for mainly preclinical assessment, due to its
short development times and low overall cost.
This book updates the latest advances in the field, with focusing on systematic
description of the technology from cell lines, cell culture conditions, vector
construction, expression strategy, current protocols, optimisation of the
procedure, and potential for clinical application. As a conclusion, the author
foresees that therapeutic biopharmaceutics will be manufactured for clinical
development using TGE technology in the near future because of its fast
development time, good protein expression, acceptable quality of product
and due to the progress which has been made in analytical methodology and
process quality control.

Update on Production of Recombinant Therapeutic
Protein: Transient Gene Expression

Published by

The objectives of this book are to summarise current TGE protocols, to describe
optimisation of the technology through the latest advances, and to explore
clinical applications of the technology. It gives the reader a good insight into
the latest development and future application of the technology platform,
including:

•
•
•
•

Jianwei Zhu

The current protocols from small to large scale for different cells.
Optimisation methods in construction designing, transfection
procedures, and cell culture conditions.
Overall quality of the product from the transient gene expression.
Future clinical application of the technology platform.

Jianwei Zhu

Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK
Telephone: +44 (0)1939 250383
Fax: +44 (0)1939 251118
Web: www.polymer-books.com

Update on Production of
Recombinant Therapeutic Protein:
Transient Gene Expression


Update on Production
of Recombinant
Therapeutic Protein:
Transient Gene
Expression
Jianwei Zhu

A Smithers Group Company
Shawbury, Shrewsbury, Shropshire, SY4 4NR, United Kingdom

Telephone: +44 (0)1939 250383 Fax: +44 (0)1939 251118




First Published in 2013 by

Smithers Rapra Technology Ltd
Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK

© 2013, Smithers Rapra Technology Ltd

All rights reserved. Except as permitted under current legislation no
partof this publication may be photocopied, reproduced or distributed
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apologise if any have been overlooked.

ISBN: 978-1-84735-976-6 (hardback)
978-1-84735-977-3 (ebook)

Typeset by Integra Software Services Pvt. Ltd.


C

ontents

Acknowledgements ......................................................................vi
Preface ........................................................................................vii
Contributors ............................................................................... ix
1.

Transient Gene Expression in Different Expression Systems .... 1
1.1

Introduction ................................................................. 1

1.2

Transient Gene Expression versus Stable
Gene Expression .......................................................... 2

1.3

Transient Gene Expression in Different Systems ........... 6
1.3.1

Mammalian Cell Systems ................................. 6

1.3.2

Plant Systems ................................................... 7

1.3.3

Insect Cell Systems ........................................... 9

1.3.4

Stem Cell Systems........................................... 11

References .......................................................................... 12
2.

Recent Advances in Transient Gene Expression Protocol..... 17
2.1

Vectors ....................................................................... 18
2.1.1 Viral Vector .................................................... 19
2.1.2 Nonviral Vectors ............................................ 23

2.2

Construction for Expression ...................................... 25
2.2.1 Promoter ........................................................ 25
2.2.2 Other Construction Components ................... 26
2.2.3 Plasmid Preparation and Quality.................... 26

iii


Update on Production of Recombinant Therapeutic Protein
2.3

2.4

2.5

Nonviral Gene Delivery ............................................. 28
2.3.1

Electroporation Methods ............................... 29

2.3.2

Chemical Methods ......................................... 30

Cell Lines used in Transient Gene Expression ............ 35
2.4.1

Human Embryonic Kidney 293 Cells ............. 40

2.4.2

Chinese Hamster Ovary Cells......................... 42

2.4.3

Other Cell Lines ............................................. 43

Current Transient Gene Expression Protocols ............ 47
2.5.1

Shake Flask Protocol for Volumes of
Normal and High Density Cell Cultures

Greater than One Litre [108] .......................... 49

2.5.2

Protocol for Large-scale Transient Transfection
in the Wave Bioreactor [71, 110].................... 51

2.5.3

High Density Large-Scale Transfection
of Mammalian Cells [109] ............................. 55

2.5.4

100 L Transient Gene Expression
Protocol [4] .................................................... 58

2.5.5

Purification of Products from Transient
Gene Expression ............................................ 61

References ......................................................................... 70
3.

Optimisation of Transient Gene Expression for
Therapeutic Protein Production .......................................... 81
3.1 Optimisation of the Transient Gene Expression
Conditions ................................................................. 86

3.2

3.1.1

Medium Optimisation .................................... 87

3.1.2

Optimisation of Transient Gene Expression
Conditions and Procedures ............................ 93

3.1.3

Construction Optimisation............................. 96

3.1.4

Coexpression of Growth Factors .................. 103

Extension of Protein Production after
Transfection ............................................................. 104
3.2.1

iv

Stable Transfection Pool ............................... 105


Contents

3.3

3.2.2

Transfection Pools with Genetic
Modification ................................................ 109

3.2.3

Plasmid Replication...................................... 112

3.2.4

Antiapoptosis ............................................... 114

Optimisation of the Technology in Other Aspects .... 119
3.3.1

Product Improvement................................... 119

3.3.2

BacMam....................................................... 121

References ........................................................................ 122
4.

Clinical Applications of the Transient Gene Expression .... 135
4.1

4.2

Quality Assessment of the Product Manufactured
by Transient Gene Expression .................................. 136
4.1.1

Glycosylation Analysis ................................. 136

4.1.2

Product Quality Consistency and
Process Reproducibility ................................ 142

4.1.3

Further Analysis of Transient
Expression Systems ...................................... 147

Clinical Development of Therapeutic
Recombinant Proteins using Transient
Gene Expression ...................................................... 149
4.2.1

Acceleration of Screening Drug Candidates
at the ‘Proof-of-Principal’ Stage ................... 149

4.2.2

Therapeutic Proteins in Clinical
Development using Other Systems ............... 150

4.3

Quality Requirements for Clinical Products ............. 151

4.4

Clinical Manufacturing of Recombinant
Therapeutic Proteins using Transient
Gene Expession........................................................ 153
4.4.1

In-process Quality Control ........................... 157

4.4.2

Product Quality Characterisation ................. 157

References ........................................................................ 158
Abbreviations ........................................................................... 161
Index ........................................................................................ 167
v


A

cknowledgements

The authors would like to extend acknowledgement to the
Biopharmaceutical Development Program of Frederick National

Laboratory for Cancer Research where the editor experienced and
accumulated knowledge of the transient gene expression technology
platform. The authors also like to thank many individuals who
directly or indirectly contributed to this book including, particularly,
Dr. Baohong Zhang for having assisted in reference listing and Tammy
Schroyer for having assisted in figures. Some of the original data were
from the presentations made by Dr. Man-shiow Jiang and Dr. Matt
Zustiak at a number of scientific symposiums.

vi


P

reface

Mammalian cells have become the dominant system for producing
70% of approved recombinant therapeutic proteins modified by
human-like post-translational modification with respect to molecular
structures and biochemical properties. There is a growing number of
therapeutic biological molecule candidates in the pipeline awaiting
preclinical and clinical evaluation. Due to its short development
times and low overall cost, transient gene expression (TGE) has been
actively pursued over the past decade to produce a wide range of
therapeutic proteins, monoclonal antibodies and vaccines, mainly
for preclinical assessment.
Over the last ten years, the remarkable progress in TGE makes
this approach attractive for supplying materials for preclinical
development and which have potential clinical applications. As
the TGE technology platform reached the 1 g/L expression level

milestone, as cell culture and transfection can be scaled up to over
100 L for production, and as products made by TGE were consistent
and reproducible, this technology platform has been widely employed
as an initial stage of biopharmaceutical development such as screening
for expression strategy in terms of construction design, molecular
candidate selection, and manufacturing products for characterisation,
which will potentially be developed for clinical applications.
This book will update the latest advances in the field. Particular
attention will be paid to systematic description of the technology
from cell lines, cell culture conditions, vector construction, expression
strategy, current protocols, transfection procedure, optimisation of
the procedure, and potential for clinical application. This book is

vii


Update on Production of Recombinant Therapeutic Protein
composed of four chapters. While TGE is used in several expression
systems that are briefly introduced in Chapter 1, this book describes
the production of biotherapeutics using the mammalian cell TGE
technology platform. In Chapter 2, current protocols are summarised
with detailed analysis of the critical steps including vector, plasmid
preparation, gene delivery methods and cell lines used. Further
optimisation of TGE procedures is described in Chapter 3 through
cell culture conditions and procedure, genetic construction and
cell line engineering. Finally, application of the TGE technology in
clinical development of biopharmaceuticals is updated, analysed,
and rationalised in Chapter 4. As a conclusion, the author foresees
that therapeutic biopharmaceutics will be manufactured for clinical
development using TGE technology in the near future because of its

fast development time, good protein expression, acceptable quality
of product and due to the progress which has been made in analytical
methodology and process quality control.
The objectives of this book are to summarise current TGE protocols,
to describe optimisation of the technology through the latest
advances, and to analyse and explore clinical applications of the
technology. A further aim is to provide up-to-date information and
reference sources for those who are working in the field to utilise in
their projects.
It is hoped that the book will be of interest to those in the field of
conducting research and development in the field of biotherapeutics,
from basic science laboratories to process and product development
in the biopharmaceutical industry. It will be a particularly useful
reference for those who are at undergraduate and graduate levels
studying biopharmaceutical development and preparing themselves
for creating the next generation of innovative biopharmaceutics.
It is also an invaluable information package for those in the
biopharmaceutical industry who are actively developing potential
new biotherapeutics through efficient methodologies.

viii


C

ontributors

Hua Jiang
Novavax, Inc., 9920 Belward Campus Drive, Rockville, MD 20850,
USA

Charles Y. Zhu
Biomedical Engineering, Rutgers University, 599 Taylor Road,
Piscataway, NJ 08854, USA
Jianwei Zhu
Shanghai Jiao Tong University and Biopharmaceutical Development
Program, National Cancer Institute at Frederick, SAIC Frederick,
Inc., 3704 Spicebush Way, Frederick, MD 21704, USA

ix



1

Transient Gene Expression in
Different Expression Systems
Charles Y. Zhu and Jianwei Zhu

1.1 Introduction
More than 130 recombinant therapeutic proteins and monoclonal
antibodies (Mab), and more than 300 non-recombinant
biopharmaceuticals (vaccines and blood products) have been
approved by the United States Food and Drug Administration (FDA)
between 1982 and 2012 [1]. Manufacturing such a huge variety of
products requires a range of production platforms. There is a growing
number of therapeutic biological molecule candidates in the pipeline
waiting for preclinical and clinical evaluation. Mammalian cells are
the favoured expression systems for clinical therapeutics since most of
the proteins require post-translational modifications, which occur in
mammalian cells, for for them to be able to carry out their therapeutic

function in the patient [2]. The traditional method of generating
these proteins routinely involves a time- and resource-consuming
process to develop stable cell lines for production. Because of its short
development time and low cost, transient gene expression (TGE) has
been actively pursued over the past decade to produce a wide range
of therapeutic proteins, Mab, and vaccines, mainly for preclinical
assessment. Technically, all the strategies used to optimise expression
in the development of a stable cell line can be used and evaluated in
TGE to assess their potential prior to committing significant resources
to create a stable cell line. Because TGE provides quick results and
costs much less than stable cell line development, it is used as the
first step to screen the gene expression strategies in terms of which
construction design and molecular candidate will be selected [3] for
potential clinical development [4].

1


Update on Production of Recombinant Therapeutic Protein
There have been many outstanding reviews and book chapters
which summarise the rapidly growing TGE technology platform
[5-8], as well as practical protocols that cover the TGE processes
from small to large scale productions [9-14]. Baldi and co-workers
[5, 14] and Pham and co-workers [6] reviewed the most commonly
used cell lines, their derivatives, and expression vectors (viral and
nonviral) for their expression and transfection conditions used
in large scale TGE. Large scale transfection up to 100 L working
volume, with a focus on human embryonic Kidney 293 (HEK293)
and Chinese hamster ovary (CHO) cells, was summarised by Baldi
[5, 14], Pham [6], Hacker [7], Geisse [11], and their co-workers

via detailed protocols and experimental notes. This book will focus
on the latest advances in this field, especially in the systematic
description of the technologies in gene delivery methods, cell
lines, current protocols, optimisation of TGE procedures, newly
developed technical aspects, and current and future applications
in clinical development.

1.2 Transient Gene Expression versus Stable
Gene Expression
TGE in mammalian cells is defined as the expression of a recombinant
protein by a gene (gene of interest [GOI]) that is introduced into a
mammalian cell and expressed only for a short defined period of
time after transfection without being stably integrated into the host
chromosomes. The TGE process, as illustrated in Figure 1.1 (left side),
starts with an established host cell bank and plasmid(s) that carry
coding sequence(s) for protein expression and through transfection
for 3-7 days. Compared to stable gene expression (Figure 1.1 right
side), the upstream process of TGE is much simpler involving only
one step of transfection of cells from an established cell bank. This

2


Transient Gene Expression in Different Expression Systems
eliminates several time-consuming operations in a stable expression
approach such as stable pool selection, single clone selection, clone
characterisation in cell growth and product expression, as well as
cell banking. In TGE, the culture is harvested in a couple of days
after transfection, followed by separation of the supernatant from
cells and cell debris. The product is recovered and purified during

downstream processing (Figure 1.1). While stable gene expression
may provide a production cell bank with homogenous seed culture,
high yield, consistent product quality, and conforms to existing
regulations, the transient expression approach offers the advantages
of less resources-demanding and short development time, which
leads to a much less effort for product production. TGE provides
a short/quick procedure to reach the point of product ‘harvest’,
beyond which further purification steps are similar for both TGE
and stable gene expression. Table 1.1 lists comparisons between
the two approaches including key factors in therapeutic protein
production: development effort, quantity of deoxyribonucleic acid
(DNA) required, expression level, product quality, production
scale, regulatory approval, and overall cost. The biggest advantages
of TGE is the short duration for development and much lower
cost when compared to stable cell line development (Table 1.1).
Furthermore, the quality of the products obtained from TGE is
suitable for preclinical assessment, drug toxicity assessment, and
potentially for early phases of human clinical trials, thus speeding
the ‘Proof of Principal’ stage in which large biopharmaceutical
companies often screen multiple drug candidates prior to advancing
into the formal development pipeline. Having listed the advantages
of using TGE, however, one has to realise that a large quantity of
high quality plasmid DNA is required and the overall expression
level of a recombinant protein or Mab is 5-10 fold lower than that
of a stable gene expression system (Table 1.1) which is obtained
after high-producer screening.

3

Table 1.1 Transient versus stable expression of recombinant
therapeutic protein in mammalian cells
Comparison
parameter

Transient expression

Stable expression

Cell line
development

Intensive effort not
usually required

Time- and resourceconsuming

Coding DNA
sequence

On a plasmid or other
vectors

Integrated into the host
genome

DNA in host cell

0-100 h, usually not
propagated through

generations

Can be maintained
throughout many
generations

DNA quantity
requirement

Large quantity
of plasmid DNA
(approximately 1mg
DNA/L cell culture)
needed

No large quantity of
plasmid DNA required

Expression level

Recombinant protein
Recombinant protein up
up to 100mg/L; Mab up to 1g/L; Mab up to 5 g/L
to 1 g/L culture volume culture volume

Productivity
(pg/c/d)

Approximately 1-10

Approximately 10-80

Product quality

Good for preclinical
assessment*

Consistent product quality
for clinical and commercial
products

Time to set up
production

Within weeks

6-12 months

Genetic selective
marker

Not needed

Needed

Current production
scale

Up to 100 L working
volume

Up to 20,000 L

Regulatory
approval

No regulatory approval
for therapeutic biologics
from mammalian cells
except for vaccines.

More than 100 biologics
have been approved
through FDA and
European Medicines
Agency (EMA) and are on
the market

Overall cost

Low

High

* For vaccine, product from transient expression may be acceptable for
clinical trials.

4

Transient Gene Expression in Different Expression Systems
Transient Gene Expression

Stable Gene Expression
Host Cell
Bank

Host Cell
Bank
Host Cell
Culture

Plasmid DNA

Product
Purification

Host Cell
Culture

Plasmid DNA

Transfection
Expression

Transfection

Harvest

Stable

Selection

Purification

Cloning

Product

Characterisation
Cell Bank

Stable Cell Line
Development

Cell Banking
Cell Culture
Expression
Harvest

Purification

Product
Purification

Product

Figure 1.1 Process flow charts for transient and stable transfection
and gene expression. The left panel is the transient gene expression
process and the right panel is stable gene expression. Both process
charts include cell culture, plasmid preparation, transfection/

expression, product recovery, and downstream purification

While TGE can be used in several expression systems that we will
briefly introduce in the following sections, the TGE technology
platforms with mammalian cells is the focus of this book. In Chapter
2, current protocols are summarised with detailed analysis of the
critical steps from vector, to plasmid preparation, gene delivery
methods, and the cell line used. Further optimisation of TGE
procedures is described in Chapter 3 including cell culture conditions
and procedure, genetic construction, and cell line engineering. Finally,

5


Update on Production of Recombinant Therapeutic Protein
application of the TGE technology in the clinical development of
biopharmaceutics is updated and rationalised. As a conclusion,
the author foresees that therapeutic biopharmaceutics for clinical
development will be manufactured using TGE technology in the
near future due to its short development time and acceptable quality
of the proteins produced, as well as due to advances in analytical
methodology and process quality control.

1.3 Transient Gene Expression in Different Systems
In order to manufacture therapeutic proteins that are needed
for research or preclinical/clinical purposes, or to control cell
differentiation as in stem cells, a gene of interest (GOI) can be
transfected into the desired target cells including mammalian, insect,
plant, and stem cells for expression. The expression can occur for a
short period of time, but does not necessarily integrate the gene into

the host chromosome and is not passed on to the next generation.
TGE in several different types of systems is briefly described in the
sections below.

1.3.1 Mammalian Cell Systems
Mammalian cells have become the dominant system for producing
over 50% of approved recombinant therapeutic proteins and in
vivo diagnostic applications because of proper protein folding and
assembly, and their post-translational modifications with respect
to molecular structures and biochemical properties are similar to
those of the human body. Recently, the productivity of mammalian
cells cultivated in bioreactors has reached 10-15 g/L for therapeutic
Mab and fragment crystallisable- (Fc-) region fusion proteins [15]
due to the improvements in cell line, expression strategies, media
optimisation, and process control. In the development pipeline, a
growing number of recombinant proteins need to be rapidly screened
to identify new candidates for clinical trials. Consequently, rapid
mammalian cell-based bioprocesses have been established with TGE,

6


Transient Gene Expression in Different Expression Systems
in which the production of recombinant proteins follows gene delivery
into cells without the establishment of a stable cell line.
The TGE technology platforms that are applied to production in
relatively small quantities (100 mg - 100 g) in mammalian cells have
been maturing in the biopharmaceutical industry. A couple of recent
outstanding examples demonstrated that TGE expression level of
Mab in HEK293E cells reached 1 g/L at the 2 L scale [16, 17] and the

production process can possibly be scaled up to 100 L. GOI can be
carried through viral or nonviral vectors and transferred into many
different cell lines [7]. Numerous gene delivery methods including
physical, for instance electroporation, and methods using chemical
reagents have been developed in mammalian cell systems over the
last 20 years. Establishment of transfection protocol, optimisation of
cell growth, development of transfection media, and choice of host
cell lines have been extensively studied. Cell lines such as HEK293,
CHO, and their derivatives have been developed as dominant hosts
in large scale (1-100 L) production. A wide range of products from
Mab [16-18], Fc fusion proteins [19], to various other recombinant
proteins [20, 21] were manufactured with the TGE system. The
major purpose of making products from mammalian system TGE is
to quickly assess candidate proteins by means of preclinical studies.
Additional details regarding notable achievements of mammalian
cell TGE will be described in subsequent chapters.

1.3.2 Plant Systems
The advantages of plant-based expression systems include high
scalability, low upstream cost, lack of human or animal pathogens,
and the capability of producing target proteins with desired structures
and biological functions through post-translational modification.
In the last few years, plants have become an increasingly attractive
production platform for recombinant pharmaceuticals including
vaccines, antibodies and other recombinant proteins [22].
TGE, as with mammalian cell systems, provides a rapid alternative
to the resources- and time-consuming generation of stably

7

Update on Production of Recombinant Therapeutic Protein
transformed plants. Using transgenic and transient expression in
whole plants or plant cell culture, a variety of recombinant subunit
vaccine candidates, and therapeutic proteins including Mab, have
been produced [23, 24]. Many plant-derived biopharmaceutical
products manufactured using approved good manufacturing
practice (GMP)-compliant processes have been in phase I to phase
III clinical trials [25]. One of them, Uplyso (a recombinant enzyme)
developed by Protalix BioTherapeutics, was approved by the FDA
in May 2012 [26]. As plant-based products have entered clinical
trials and even the market, there has been increased emphasis on
manufacturing under current GMP guidelines, and the preparation
and presentation of regulatory packages to the relevant government
agencies [25]. Eukaryotic protein processing coupled with
reduced production costs and low risk for mammalian pathogen
contamination and other impurities have led many to predict that
agricultural systems may offer the next wave for pharmaceutical
product production [23].
TGE in plants has been quite successful. The advantages of these
plant cell factory systems include ease of manipulation, speed,
low cost, high protein yield, scalability and tight control of both
upstream and downstream processing during manufacturing.
When DNA is delivered into a plant cell, only a tiny proportion
will become integrated into the host chromosomes and episomal
DNA molecules can remain transcriptionally competent for several
days. This transient expression does not depend on chromosomal
integration and is not affected by position effects. Expression from
extra-chromosomal transgenes can be detected in as little as 3 h
after DNA delivery, reaches the maximum between 18 and 48 h,

and persists for ten days. As stated by Pogue and co-workers
[23], the time taken by transient systems to produce milligrams of
product can be as short as two weeks and the production of gram
quantities of product may take only a few weeks. These timeframes
are much shorter than those required to transfect, select, establish
and characterise mammalian cells, transgenic animals, or traditional
plant-based systems.

8


Transient Gene Expression in Different Expression Systems
To date, there have been many examples of recombinant proteins
synthesised using plant TGE systems entering clinical trials and
one is even a marketed product [24-26]. Recent examples utilising
TGE technology are the personalised therapeutic vaccine for nonHodgkin lymphoma based on recombinant single-chain variable
fragment antibodies and an H5N1 pandemic influenza vaccine based
on the production of virus-like particles (VLP) [27, 28]. Both were
transiently produced in Nicotiana benthamiana plants and are in
clinical trials [27, 28].

1.3.3 Insect Cell Systems
While mammalian systems are the most desirable for biotherapeutic
manufacturing to deliver fully functional proteins, alternatives
including insect cell expression systems became economically valuable
due to high expression, simple culture conditions, and relatively
low capital investment. In addition, insect cell culture substantially
reduces the risk of human virus contamination that causes safety
concerns in human clinical applications. However, a safety issue with
residual viral DNA in the final products may have to be addressed

if the Baculovirus-insect cell system is used. Further, differences
between insects and humans in glycosylation may cause side immune
responses in humans. With the first licensed human Papillomavirus
vaccine produced with the Baculovirus-expression system approved
by the EMA in 2007 [40] and by the FDA in 2009 [41], this system
is being developed as a platform for recombinant vaccine production
with confidence by the biopharmaceutical industry [42].
The Baculovirus expression vector system (BEVS) is based on the
introduction of a foreign gene into the nonessential genome region
for viral replication via homologous recombination with a transfer
vector containing target gene. The resulting recombinant Baculovirus
lacks one nonessential gene (polh, v-cath, chiA and so on) which has
been replaced with a foreign gene encoding heterologous protein. The
GOI can be then expressed in cultured insect cells. Sf-9, Sf-21, and

9


Update on Production of Recombinant Therapeutic Protein
other cells, derived from Spodoptera frugiperda, are widely used for
recombinant protein production using the BEVS. Protein expression
in insect cells allows post-translational modifications (signal peptide
cleavage, phosphorylation, lipid modification, and glycosylation) as
well as structural folding of proteins that are required for biological
activity. Higher order protein structures can also be produced in
this expression system, such as enzyme complexes, VLP that selfassemble from structural viral proteins, and viral-derived gene
delivery vehicles [29]. The first recombinant vaccine on the market
(Cervarix [GlaxoSmithKline]) is a virus-like particle-based vaccine
directed against cervical cancer [30]. Baculoviral-insect expression
systems can reach expression levels up to 1 g/L of the protein of

interest. Insect cell cultures (Sf9, Sf21, and others) are easy to grow
and to be maintained as either adherent or suspension cultures at
27 °C, and can be easily scaled-up for production. Baculoviruses
infect only insects and are nonpathogenic to humans.
Besides Baculovirus infection as a gene delivery system, suitable
expression plasmids and key reagents are also available from
commercial sources. Farrell and Iatrou [31]described the production
of moderate to good yields of several proteins on a small scale in High
Five Cells in suspension following lipofection-mediated gene transfer.
One recent publication showed that Sf-9 cells were transfected with
polyethylenimine (PEI)/plasmid DNA in a TubeSpin bioreactor. The
system demonstrated that by five days post-transfection, 58% of
the cells were green fluorescent protein-positive and by seven days
post-transfection, the product of fusion protein tumor necrosis
factor receptor as an Fc fusion concentration reached 42 mg/L [32].
Facilitated by PEI, this non-lytic plasmid-based method appears
simple, efficient, and cost-effective for TGE of recombinant protein
in insect cells cultivated in serum-free suspension mode [32]. It is a
valuable alternative to the BEVS for rapid, scalable, and high-yielding
recombinant protein production and for the generation of stable Sf-9
cell lines [33].
Optimised protocols for transient transfection in Sf and High Five
cells using lipofection with Gene Juice (Novagen, Merck) and

10


Transient Gene Expression in Different Expression Systems
Cellfectin (Invitrogen) as well as PEI-mediated transfection were
reported by Geisse [8]. A detailed protocol using Baculovirusmediated and plasmid-driven expression of a candidate gene was

presented by Buchs and co-workers [34].

1.3.4 Stem Cell Systems
Human embryonic stem (hES) cells have raised great expectations in
regenerative medicine because of their potential of being an unlimited
source of cellular materials. To direct and control their differentiations
for biomedical applications, temporary expression of the regulatory
genes without permanently altering the genomes would be essential
and could be superior to long-term constitutive transgene expression.
For purposes other than producing recombinant therapeutic proteins
and vaccines in mammalian, plant, and insect cells, TGE in stem cells
is designed to regulate and control cell differentiation. In addition,
it might also be possible to use transient expression of certain
self-renewal related genes to stimulate stem cell division without
differentiation, thus expanding hES cells and obtaining clinicallyrelevant amounts of the cells as a source for the development of
cell-based therapeutic products [35]. For developmental biology
research in investigating the signalling pathways, transient expression
within a desired time window would be useful and crucial for the
proliferation and differentiation of hES cells.
The most common method of generating transiently or stably DNAtransfected mouse embryonic stem cells is electroporation [36]. Great
efforts have been made to develop gene transfer vectors that can
efficiently mediate transgene expression in hES cells with nonviral
vectors or methods offering nonintegrative strategies for transient
expression [37] or with helper-dependent adenoviral vectors [38].
The development of non-integrating lentiviral vectors provides a
novel tool for efficient transient gene expression in primary stem
cells and hematopoietic and lymphoid cells. One recent report states
that the ‘Sleeping Beauty’ transposon system has been developed and
validated for ex vivo gene delivery to stem cells, including T-cells for

11


Update on Production of Recombinant Therapeutic Protein
the treatment of lymphoma. This vector combines the advantages of
viruses and naked DNA [39]. With all the progress in developmental
research, many clinical trials utilising hES cells are currently being
conducted [43].

References
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