Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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EGF epidermal growth factor

EMEA European Medicines Agency

EPO erythropoietin

FDA Food and Drug Administration

FIX blood coagulation factor IX

FSH follicle-stimulating hormone

FVII blood coagulation factor VII

FVIII blood coagulation factor VIII

G-CSF granulocyte colony-stimulation factor

GM-CSF granulocyte-macrophage colony-stimulation factor

IFN interferon

IGF insulin-like growth factor

IgG immunoglobulin G

IL interleukin

IND investigational new drug application

LH luteinising hormone

mAb monoclonal antibody

M-CSF macrophage colony-stimulation factor

PDGF platelet-derived growth factor

PEG polyethylene glycol

TGF transforming growth factor

TNF tumor necrosis factor

TNKase tenecteplase (commercial second-generation tPA)

tPA tissue plasminogen activator

VEGF vascular endothelial growth factor

Chapter 17

ATP Adenosine triphosphate

BHK baby hamster kidney cell line

CD cluster of differentiation

cDNA complementary DNA

CDR Complementarity determining regions

CHO Chinese hamster ovary cell line

CMC carboximethylcellulose

DMSO Dimethylsulfoxide

DNA Deoxyribonucleic acid

DO dissolved oxygen

ELISA enzyme linked immunosorbent assays

Fc functional fragments

Fv variable fragments

HACA Human Anti-Chimeric Antibody

HAMA Human Anti-Murine Antibody

HAT aminopterin, hypoxanthine, and thymidine

HEK-93 human embryonic kidney cell line

HGPRT hypoxanthine phosphoribosyl transferase

Ig Immunoglobin

IgA Immunoglobin class A

IgD Immunoglobin class D

xxx Animal Cell Technology

IgE Immunoglobin class E

IgG Immunoglobin class G

IgM Immunoglobin class M

IL Interleukin

a Volumetric oxygen transfer coefﬁcient

KLH keyhole limpet haemocyanin

mAb Monoclonal antibody

PEG polyethylene glycol

S-S Disulﬁde bridge

TK thymidine kinase

tRNA transcript of the RNA

Chapter 18

BTV Blue tongue virus

cccDNA Covalently closed circular DNA

CLPs Core-like particles

CTL Cytotoxic T-lymphocytes

dDNA Double-strand DNA

dRNA Double-strand RNA

HBsAg hepatitis B virus surface antigen

HBV Hepatitis B virus

mRNA Messenger rNA

+mRNA Positive strand messenger RNA

PPV Porcine parvovirus

RNA- Complementary negative RNA strand

RNA+ Genomic RNA

-sRNA Negative single-strand RNA

+sRNA Positive strand RNA

RNAs Genomic RNA

ssRNA Single-stranded RNA

MOI Multiplicity of infection

TOI Time of infection

VLPs Virus-like particles

VP1 Viral protein 1

VP2 Viral protein 2

VP3 Viral protein 3

VP4 Viral protein 4

VP5 Viral protein 5

VP6 Viral protein 6

VP7 Viral protein 7

Chapter 19

AcMNPV Autographa californica NPV

AfMNPV Anagrapha falcifera nucleopoliedrovirus

BmMNPV Bombyx mori NPV

BV budded virus

DIP Defectives Interfering Particles mutants

DNA Deoxyribonucleic acid

Abbreviations and Symbols List xxxi

EMBRAPA Brazilian Agricultural Research Corporation

FBS fetal bovine serum

FP Few Polyedra mutants

GmMNPV Galleria mellonella NPV

GMO genetically modiﬁed organisms

GV Granulovirus

HaSNPV Helicoverpa armigera NPV

HzSNPV Helicoverpa zea nucleopoliedrovirus

IEA Institute of Agriculture

LdMNPV Lymantria dispar nucleopoliedrovirus

MNPV multiple NPV type

MOI multiplicity of infection

NPV Nucleopolyedrovirus

OB occlusion body

ODV occlusion-derived virus

OpMNPV Orgyia pseudotsugata NPV

Sf9 and Sf21 Spodoptera frugiperda cell line

SfMNPV Spodoptera frugiperda NPV

SNPV simple NPV type

TN-368 Trichoplusia ni cell line

TnMNPV Trichoplusia ni NPV

Chapter 20

3G5 membrane ganglioside related to pericytes identiﬁcation

BMP-12R receptor to bone morphogenetic protein isoform 12

Brachyury transcription factor related to embryo mesoderm determination

CBF core-binding factor; protein family involved in cell differentiation

processes

CD3/cCD3 protein complex tyrosine kinase associated with T cell receptor (TCR);

detected on cell surface or intracytoplasm (c)

CD4 TCR co-receptor; binds to MHC-II (major histocompatibility complex –

class II)

CD7 transmembrane protein linked to PI-3 (phosphoinositol-3) kinase;

cellular function not thoroughly clariﬁed

CD8 TCR co-receptor; binds to MHC-I

CD10 metalloproteinase Zn-bound

CD11b integrin Æ

; associated with CD18 (integrin 

); CD54 ligand; binds

complement system iC3b and ECM (extracellular matrix) proteins; also

known as Mac-1

CD11c integrin Æ

; associated with CD18; ﬁbrinogen ligand; also known as

Mac-3

CD13 aminopeptidase N (metalloproteinase Zn-bound)

CD14 LPS (lipopolysaccharide) receptor

CD16/32 FcªRII/III (receptor II/III to Fcª)

CD19 B cell receptor co-receptor linked to PI-3 kinase; forms complex ligands

with CD21 (C3b receptor) and CD81

CD25 IL-2 Æ chain receptor

CD29 

integrin ; forms a complex with CD49/Æ-integrin

CD31 PECAM/platelet-endothelial cell adhesion molecule

xxxii Animal Cell Technology

CD33 gp67/glycoprotein 67 expressed by myelomonocytic progenitors;

function not thoroughly yet deﬁned

CD34 gp105-120; binds to CD62L (L-selectina); antigen essential to identify

human stem cells; function not yet completely known

CD35 C1 and C4b complement protein system receptor

CD38 NAD glycohydrolase; expression related to cell proliferation increase

CD41 integrin/gpIIb Æ

IIb

chain; works with CD61 (gpIIIa), binding to

ﬁbrinogen, vWF and trombospondin

CD44 H-CAM (Hermes cell adhesion molecule); binds to hialuronic acid and

promotes mild leukocyte interaction

CD45 LCA/leukocyte common antigen; tyrosine phosphatase that reinforces T

and B lymphocyte receptor signaling

CD45RA isoform of CD45 containing the exon A; expressed on naı

ve T, B cells

and monocyte subpopulations

CD49 Æ chain integrin; involved in cell interactions

CD54 ICAM-1 (intercellular adhesion molecule); this binds to CD11a-b/CD18

complex

CD56 NCAM (neural cell adhesion molecule); good marker for NK cells

CD60 9-O-acetyl GD3; surface ganglioside involved in receptor complexes

during cell signaling

CD62L L selectin; related to co-binding with CD34 and interactions to

endothelia

CD64 FcªR1/receptor 1 to Fc gamma

CD66a BGP (biliary glycoprotein); member of carcino-embryonary antigen

(CEA) family

CD71 receptor to transferrin

CD73 ecto-5’-nucleotidase; involved on nucleotide dephosphorylation

CD81 TAPA-1 (TAPA-1 – target for antiproliferative antigen-1); associated

with CD19 and CD21

CD87 plasminogen receptor activator

CD90 also known Thy-1; T-lymphocyte marker involved in cellular adhesion

CD100 semaphorin subtype; function not completely deﬁned

CD104 4 integrin chain; associated with CD49 and binds to laminin

CD105 endoglin; TGF- co-factor

CD106 VCAM-1 (vascular cell adhesion molecule); this binds to VLA-4 (very

late antigen)

CD110 trombospondin receptor

CD117 SCF (stromal or stem cell factor) receptor; mostly known as cKit

CD122 -chain of IL-2receptor

CD123 Æ-chain of IL-3 receptor

CD125 Æ-chain of IL-5 receptor

CD127 Æ-chain of IL-7 receptor

CD135 FLT-3/Flk-2; also known as Flt-3L (fms-related tyrosine kinase ligand)

receptor

CD144 VE-cadherin; adhesion molecule related to endothelial cells

CD146 MCAM (mesenchymal cell adhesion molecule); also known as MUC-18,

S-endo or MEL-CAM

CD166 gp37; molecule involved in adhesion between lymphocytes and thymic

epithelial cells

Abbreviations and Symbols List xxxiii

CD203c ecto-enzyme ectonucleotide pyrophosphatase/phosphodiesterase 3 (E-

NPP3); mostly expressed in basoﬁles

CD235a glycophorin A; membrane protein expressed in red blood cells

Col collagen

cTn cardiac throponin

CXCR4 chemokine receptor CXC 4

EPO-R receptor of erythropoietin

F4/80 membrane glycoprotein related to macrophage activity

FGF-R ﬁbroblast growth factor receptor

GM-1 membrane ganglioside related to nervous cells

GM-CSFR granulocyte/monocyte - colony stimulation factor receptor

Gr-1 membrane antigen expressed mainly on granulocytes

HLA-DR human leukocyte antigen – DR; analogous to mice MHC

HLA-II HLA class II

IgE-R immunoglobulin E receptor

IL-5R receptor of IL-5

KDR also known as ﬂk-1 and VEGF-R2 (vascular/endothelial growth factor

receptor 2)

Lin abbreviation for lineage; related to hematopoietic cell lineages, namely:

CD3/4/8/11b/19/56, Gr-1 and Ter 119 (mice) ou CD235a (human)

LPL L lipoprotein

M-CSFR monocyte - colony stimulation factor receptor

MHC myosin heavy chain

MHC-II MHC class II

MMCP mouse mast cell protease

MMP metalloproteinase

NG2 proteoglycan heparan-sulfate related to pericytes activity

PDGF platelet-derived growth factor

PPAR peroxisome proliferator-activated receptor gamma

RANK receptor activator of NF-kappa 

Sca-1 stem cell antigen 1

SM22 smooth cell protein; structurally related to calponin, both actin and

tropomyosin ligands

SMA smooth muscle actin

SSEA-I stage-speciﬁc embryonic antigen; characterized as a glycosphingolipid

similar to Ii (MHC invariant chain)

Stro-1 stromal cell surface antigen, called stromal antigen 1

Ter-119 mice red blood cell marker

Tie-2 receptor tyrosine kinase related to endothelial cells activity

VEGF-R1 receptor 1 of VEGF

VEGF-R3 receptor 3 of VEGF

vWF Von Willebrand factor. Protein factor involved in hemostasis

Chapter 21

DNA deoxyribonucleic acid

RNA ribonucleic acid

iRNA interference RNA

AAV adeno-associated virus

HSV herpes simplex virus

xxxiv Animal Cell Technology

Foreword

In vitro culture of animal cells originated towards the end of the nineteenth century as a

collection of experimental procedures used to isolate and maintain viable cells from organs

of diverse animals, at least for a few days. From these modest origins, and throughout a

little more than 100 years, animal cell culture has evolved into a modern technology based

on scientiﬁc and engineering principles. As a result of such developments, animal cell

culture is nowadays used successfully for the production of vaccines and recombinant

glycoproteins. In addition, applications of animal cell culture now underpin new and

fascinating ﬁelds, such as organ transplants, cell and gene therapy, in vitro toxicology and

physiology, tissue analogs, production of biopesticides, bioelectromechanical devices, and

nanobiotechnology. Interestingly, of the more than 500 biopharmaceutical products that

are being evaluated in clinical trials, about half are produced by animal cell culture. Such

facts underline the importance of this technology. Furthermore, the products derived from

animal cells have an annual market of more than ten thousand million dollars with

impressive growth rates. Nonetheless, far more important than their economic value is the

impact that such products have made on human health, increasing the quality of life.

The ﬁrst therapeutic product generated by animal cells, tPA, was approved in 1986. In

1987, the ﬁrst therapeutic monoclonal antibody, OKT3, was also approved, although this

protein was obtained from the ascites ﬂuid produced in vivo. It is notable that in merely

two decades, close to 50 products produced by in vitro animal cell culture have now been

approved. From these, approximately 60% are monoclonal antibodies. Such an achieve-

ment was the result, to a large extent, of the extraordinary advances that animal cell culture

technology has experienced since the 1980s. Since then, cell and product concentrations

have increased between one to two orders of magnitude, yielding bioprocesses that can

routinely attain more than 50 x 10

cell/mL and 5 g/L of product. Likewise, due to

advances in cell line selection and new expression systems, speciﬁc cell productivities above

100 pg/cell-d are now a reality. The problem of cell fragility, that drew the attention of

researchers during the 1980s and 90s, was adequately overcome. During the last two

decades, an important effort was focused on cell metabolism and physiology. Such under-

standing allowed, among other things, a better deﬁnition of culture media. Consequently, it

was possible to eliminate undeﬁned and undesirable components, such as fetal bovine

serum, and chemically deﬁned media became widely used. Knowledge of cell death

processes resulted in novel culture schemes and molecular strategies to contend with this

problem. With the improvement in analytical methods and comprehension of protein post-

translational modiﬁcations, a considerable advancement was possible in understanding the

relation between the bioprocess and the quality of the glycoproteins produced. All these

developments permitted the practical, efﬁcient, and robust culture of animal cells in

bioreactors at scales larger than 10 m

for consistently producing safe and effective

biopharmaceuticals.

In the present book, Animal Cell Technology: From Biopharmaceuticals to Gene

Therapy, editors Drs Leda R. Castilho, A

ngela M. Moraes, Elisabeth F. Pires Augusto and

Michael Butler have integrated the background knowledge and developments of the last 20

years that have converted the culture of animal cells into a vigorous and important scientiﬁc

ﬁeld. Accordingly, basic themes are reviewed in the book, including cloning and expression

Foreword xxxv

of heterologous proteins, culture media, metabolism, cell growth and death. Technological

aspects are also examined, including the design, monitoring and control of bioreactors, and

processes for separation of cells and products. Topics of major relevance, such as the

quality control of products, regulatory issues and intellectual property, are included in

several chapters. Finally, some of the most important products and applications of animal

cell culture, including recombinant glycoproteins, monoclonal antibodies, viral vaccines,

bioinsecticides, and gene and cell therapies, are reviewed in detail.

This book includes contributions of distinguished researchers from Canada and Ibero-

America, in particular, from Brazil, Argentina, Cuba, Portugal and Uruguay. These authors

impart a special character to the book, since it reﬂects the global impact that the discipline

has acquired, and provides the vision and topics that are of interest to countries of this

region. It also represents an additional effort, started in 2004 by Drs Leda R. Castilho and

Ricardo Medronho during the ‘‘ﬁrst Latin-American Seminar on Cell Culture Technol-

ogy’’, to strengthen the relationship between Ibero-American technologists and scientists

working in animal cell culture.

The reader of the present book will be able to recognize that there still exist major

technological and scientiﬁc challenges, as well as promising opportunities, in the ﬁeld of

animal cell culture. It is possible to anticipate important changes in paradigms that will

determine the future of the ﬁeld. Twenty years after the ﬁrst products were approved,

many patents are now starting to expire. This has brought to a central stage the discussion

of the concept of biogenerics, not only from a commercial perspective, but also from a

technical and regulatory standpoint. Such a topic should be dealt with from a scientiﬁc

approach and particularly supported by detailed analytical characterization of the mole-

cules and their therapeutic efﬁcacy, aspects in which there has been outstanding recent

progress. Progress in this area will certainly contribute to defeat dogmas such as ‘‘the

process determines the product’’, which in turn will open new possibilities for developing

improved and novel bioprocesses that efﬁciently yield safe biopharmaceuticals at accessible

prices to a larger number of patients. Advances in genomics and proteomics should

contribute in bringing to the market an increasingly large number of products, derived

from animal cell culture, for treating an increasing number of diseases. Again, new techno-

logical, medical and regulatory paradigms will most likely be seen with the advent of cell

and gene therapies. Monoclonal antibodies, that are required in very large amounts, are

ﬁnally fulﬁlling the therapeutic promises generated in the mid-70s, and with this, new

manufacturing challenges can be foreseen. The race to develop bioprocesses that can yield

concentrations above 10 g/L has started. It is likely that, by integrating the knowledge

generated in the ﬁeld during the last two decades, such a goal will be reached in a short

time. A few examples that will contribute to reaching such a goal include novel operation

and control systems, new culture media supplemented with hydrolyzed proteins from plant

or yeast, and metabolic engineering of novel cell lines. New paradigms will also be

established in the puriﬁcation of biopharmaceuticals. Accordingly, it is likely that soon we

will see the application of unit operations that are presently uncommon for the ﬁeld, such

as precipitation, crystallization, and extraction, whereas the importance of common but

costly methods used nowadays, such as chromatography, will diminish in large-scale

operations. This should bring a simpliﬁcation and cost reduction of bioprocessing. On the

other hand, regulatory requirements will most likely affect and deﬁne the future of animal

cell culture. A clear example of this is the trend to use disposable equipment and

instrumentation, which should represent a fertile area for innovation.

It is clear that animal cell culture will remain an exciting and highly dynamic ﬁeld in the

years to come, and that the products generated by this technology will beneﬁt an increasing

number of people. Accordingly, the book Animal Cell Technology: From Biopharmaceu-

xxxvi Animal Cell Technology

ticals to Gene Therapy should be an important ally for technologists and scientists

interested in the topic.

Octavio T. Ramı´rez

Instituto de Biotecnologı´a, UNAM

Cuernavaca, Me´xico

Cover Photo Acknowledgement

Photo 1

Clumps of suspension-adapted CHO cells stained with ethidium bromide and acridine

orange, observed under a ﬂuorescence microscope. Photo taken by Dr. Rodrigo C. V.

Pinto, Cell Culture Engnieering Laboratory, Federal University of Rio de Janeiro, Brazil.

Photo 2

Adherent CHO cells. Photo taken by Dr. Rodrigo C. V. Pinto, Cell Culture Engnieering

Laboratory, Federal University of Rio de Janeiro, Brazil.

Photo 3

Laboratory-scale stirred-tank bioreactor for animal cell cultiavaion. Photo taken by Dr.

Rodrigo C. V. Pinto, Cell Culture Engnieering Laboratory, Federal University of Rio de

Janeiro, Brazil.

Photo 4

Inner surface of an industrial-scale stirred-tank bioreactor for animal cell cultivation. Photo

taken by Dr. Ernesto Chico, Center of molecular Immunology, Cuba.

Foreword xxxvii

Intr oduction to animal cell

technology

Paula Marques Alves, Manuel Jose

Teixeira Carrondo, and

Pedro Estilita Cruz

1.1 Landmarks in the cultur e of animal cells

Despite the dominance of animal cell culture in the production of

biopharmaceuticals in recent times, this technology was not consolidated

into standardized large-scale bioprocesses until the 1990s. Nevertheless,

the ﬁrst experience with animal cell culture can be traced back to the

beginning of the 20th century. By the use of the hanging drop technique

and frog heart lymph, Ross Harrison, at Yale, tried between 1906 and 1910

to elucidate how the nervous ﬁber is originated (Witkowski, 1979). He

considered three hypotheses: (i) in situ formation from the nerve sheath;

(ii) preformed protoplasmic bridges; or (iii) as a result of the nerve cell

growth itself. When Harrison demonstrated the validity of the third

hypothesis, he also conﬁrmed the cell as the primary developing unit of

multicellular organisms.

An early pioneer of cell culture was the French surgeon Alexis Carrel,

who won the Nobel prize in Medicine in 1912 for his research at the

Rockefeller Institute (Spier, 2000). Harrison was, above all, the inventor of

analytical solutions, while Carrel, with his extensive clinical practice

experience, sterility concerns, and capacity to develop appropriate culture

media and culture ﬂasks, created the change in the technological paradigm

that led to the start up of animal cell technology. By careful manipulation,

Carrel insured the maintenance of chicken embryo cells for several decades

in culture.

Spier (2000) lists some essential differences between cells in an organism

(in vivo) and cells in culture (in vitro), particularly the following;

(i) Tissues are three-dimensional, while cell cultures are of zero dimen-

sion (monodispersed in suspension culture) or two-dimensional

(monolayer growth). However, some culture techniques exploit

three-dimensional systems (Alves et al., 1996; Powers et al., 2002).

(ii) In tissues, cells are subject to tension and compression, but not when

in culture, with the exception of artiﬁcial organs.

(iii) In tissues, lymphokines and chemokines vary in proportion and

concentration to allow ﬂuctuations of short (cardiac rhythm), med-

ium (daily), and long duration (life cycle). However, in culture these

parameters normally do not vary.

(iv) The mechanisms for cellular differentiation control in tissues and in

culture are distinct.