Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy
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12.4.3 Separation processes based on differences in electrical charge 309
12.4.4 Separation processes based on differences in hydrophobicity 313
12.4.5 Separation processes based on specificity of ligands 314
12.4.6 Other developments 319
12.5 Conclusions 323
References 324
13 Quality control of biotechnological products 329
Marina Etcheverrigaray and Ricardo Kratje
13.1 Introduction 329
13.2 Production of recombinant proteins 331
13.2.1 Control of starting materials 331
13.2.2 Quality control of cell banks 333
13.3 Control of the production process 334
13.3.1 Cultures 334
13.3.2 Purification 335
13.4 Product control 335
13.4.1 Characterization and specification 335
13.4.2 Protein content 336
13.4.3 Amino acids analysis (identification and/or protein content) 336
13.4.4 Protein sequencing (identification) 337
13.4.5 Peptide mapping 337
13.4.6 Electrophoresis 337
13.4.7 Carbohydrate determination 340
13.4.8 Potential impurities and contaminants of biotechnological products 340
13.5 Bioassays 341
13.5.1 Bioassay types 342
13.5.2 In vitro bioassays 343
13.5.3 Experimental design 344
13.5.4 Statistical analysis 345
References 345
14 Regulatory aspects 349
Maria Teresa Alves Rodrigues and Ana Maria Moro
14.1 Introduction 349
14.2 Good Manufacturing Practices and quality assurance 350
14.3 Regulatory agencies 351
14.4 Harmonization 352
14.5 Premises 353
14.5.1 Clean rooms 353
14.5.2 Biosafety 354
14.6 Cell banks 355
14.6.1 Cell bank qualification 355
14.7 Validation 358
14.7.1 General aspects 358
14.7.2 Biological products 360
14.8 Stability 362
14.9 Clinical trials 362
14.9.1 Preclinical studies 363
x Animal Cell Technology
14.9.2 Clinical studies 364
14.10 Biogenerics or biosimilars 365
References 367
15 Intellectual property 373
Ana Cristina Almeida Mu
¨
ller and Leila Costa Duarte Longa
15.1 Introduction 373
15.2 The biotechnology sector 373
15.3 Ethical and moral aspects of research involving genetic engineering 374
15.4 Basic concepts of patentability 376
15.4.1 Discovery versus invention 376
15.4.2 Requirements for the patentability of inventions 377
15.5 Patentable materials 382
15.6 Industrial property and technology transfer offices 384
15.7 Patent and technology transfer specialists 386
15.8 Conclusions 388
References 388
16 Recombinant therapeutic proteins 389
Maria Candida Maia Mellado and Leda dos Reis Castilho
16.1 Introduction 389
16.2 Main therapeutic proteins 389
16.2.1 Cytokines 390
16.2.2 Hematopoietic growth factors 392
16.2.3 Growth factors 392
16.2.4 Hormones 393
16.2.5 Therapeutic enzymes 393
16.2.6 Blood coagulation factors 398
16.2.7 Antibodies 399
16.3 Economic aspects 400
16.4 Challenges and future perspectives 402
16.4.1 Formulation and delivery of biopharmaceuticals 402
16.4.2 Characterization of biopharmaceuticals 404
16.4.3 Alternative expression systems 404
16.4.4 Second-generation biopharmaceuticals 405
References 406
17 Monoclonal antibodies 409
Wirla M.S.C. Tamashiro and Elisabeth F.P. Augusto
17.1 Introduction 409
17.2 Antibodies 411
17.3 Production of monoclonal antibodies 415
17.3.1 Step 1: Immunization 415
17.3.2 Step 2: Fusion and selection of secreting hybridomas 416
17.3.3 Step 3: Hybridoma cloning 417
17.3.4 Step 4: Definition of the isotype of monoclonal
antibodies obtained 417
17.3.5 Step 5: Follow-up/later developments 417
17.4 Production of recombinant antibodies 418
Contents xi
17.4.1 Humanized antibodies 420
17.4.2 Human antibodies 421
17.5 Production systems 425
17.5.1 Cell lines 426
17.5.2 Basic conditions for in vitro cultivation 427
17.5.3 Cell metabolism 428
17.5.4 Bioreactors and operation mode 429
References 430
18 Viral vaccines: concepts, principles, and bioprocesses 435
Isabel Maria Vicente Guedes de Carvalho Mello, Mateus Meneghesso da
Conceic¸a
˜
o, Soraia Attie Calil Jorge, Pedro Estilita Cruz, Paula Maria
Marques Alves, Manuel Jose
´
Teixeira Carrondo, and Carlos Augusto Pereira
18.1 Introduction 435
18.2 Viral replication 436
18.2.1 Adsorption 437
18.2.2 Internalizing and unwrapping the viral particle 437
18.2.3 Structure and organization of viral genomes 437
18.2.4 Production and maturation of viral particles 442
18.3 Production of viral particles by cell culture 442
18.4 Strategies for the production of virus-like particles 447
18.4.1 Advantages of VLPs 448
18.4.2 VLP production technology 448
18.4.3 VLP composition 449
18.4.4 VLP production processes 450
18.5 Development of viruses for DNA vaccines 451
18.6 Perspectives for the evolution of viral vaccine production 452
References 455
19 Bioinsecticides 459
Ma
´
rcia Regina da Silva Pedrini and Ronaldo Zucatelli Mendonc¸a
19.1 Introduction 459
19.2 Baculovirus as a bioinsecticide: mechanism of action 460
19.3 Animal cell cultures for baculovirus production 463
19.4 Effect of culture medium, cell line, and virus isolate on biopesticide
production 463
19.5 Polyhedra virulence and characteristics 466
19.6 Production of viral mutants in cell culture 467
References 470
20 Cell therapies and stem cells 475
Hamilton da Silva Jr and Radovan Borojevic
20.1 Introduction 475
20.2 Primary material 476
20.2.1 Stem and mature cells 477
20.2.2 Tissue environment and specific niches 484
20.3 Applications 485
20.3.1 Bioexpansion and biostorage 485
20.3.2 Bioengineering 486
xii Animal Cell Technology
20.4 Conclusions and perspectives 487
References 487
21 Gene therapy 489
Ce
´
lio Lopes Silva, Karla de Melo Lima, Sandra Aparecida dos Santos, and
Jose
´
Maciel Rodrigues Jr
21.1 Introduction 489
21.2 Gene therapy 489
21.3 Vectors used in gene therapy 491
21.3.1 Viral vectors 491
21.3.2 Synthetic vectors: plasmid DNA 493
21.4 Principles of gene therapy 497
21.4.1 Replacement or correction of a mutant gene 497
21.4.2 Introduction of a heterologous gene 498
21.4.3 Gene inactivation 498
21.5 Gene therapy and clinical studies 498
21.5.1 The first gene therapy product 501
21.6 Perspectives 502
References 502
Appendix 505
Case study: Mathematical modeling of the monoclonal
antibody anti-TNP (trinitrophenyl) 505
Index 507
Contents xiii
Contributors
Paula Marques Alves, Instituto de Biologia Experimental e Tecnolo´ gica (IBET-ITQB),
Portugal
Paola Amable, Cell Culture Engineering Laboratory, PEQ/COPPE, Federal University of
Rio de Janeiro, Brazil
Elisabeth de Fatima Pires Augusto, Institute of Technological Research of the State of Sa
˜
o
Paulo, Brazil
Manuel Filgueira Barral, Fundac¸a
˜
o Santo Andre´, Brazil
Mariela Bollati-Fogolı
´n
, Cell Biology Unit, Institut Pasteur de Montevideo, Uruguay
Radovan Borojevic, Institute for Biomedical Sciences, Federal University of Rio de
Janeiro, Brazil
Sonia Maria Alves Bueno, Department of Biotechnological Processes, School of Chemical
Engineering, State University of Campinas, Brazil
Michael Butler, Department of Microbiology, University of Manitoba, Canada
Manuel Jose
´
Teixeira Carrondo, Instituto de Biologia Experimental e Tecnolo´ gica (IBET-
ITQB), Portugal
Leda dos Reis Castilho, Cell Culture Engineering Laboratory, PEQ/COPPE, Federal
University of Rio de Janeiro, Brazil
Mateus Meneghesso da Conceic¸a
˜
o, Laboratory of Viral Immunology, Butantan Institute,
Brazil
Ernesto Chico, Center of Molecular Immunology, Cuba
Marcelo A. Comini, Centre of Biochemistry, Heidelberg University, Germany
Pedro Cruz, Instituto de Biologia Experimental e Tecnolo´ gica (IBET-ITQB), Portugal
Marina Etcheverrigaray, Facultad de Bioquı´mica y Ciencias Biolo´ gicas, Universidad
Nacional del Litoral, Argentina
Alvio Figueredo, Center of Molecular Immunology, Cuba
Adriana Lages Lima Galesi, Department of Biotechnological Processes, School of Chemi-
cal Engineering, State University of Campinas, Brazil
Soraia Attie Calil Jorge, Laboratory of Viral Immunology, Butantan Institute, Brazil
Ricardo Kratje, Facultad de Bioquı´mica y Ciencias Biolo´ gicas, Universidad Nacional del
Litoral, Argentina
Patrı
´
cia Le
´
o, Institute of Technological Research of the State of Sa
˜
o Paulo, Brazil
Karla de Melo Lima, Nanocore Biotecnologia Ltda, Brazil
Leila Costa Duarte Longa, Fundac¸a
˜
o Oswaldo Cruz, Brazil
Ricardo de Andrade Medronho, Chemical Engineering Department, School of Chemistry,
Federal University of Rio de Janeiro, Brazil
Maria Candida Maia Mellado, Instituto de Biologia Experimental e Tecnolo´ gica (IBET-
ITQB), Portugal
Isabel Maria Vicente Guedes de Carvalho Mello, Laboratory of Viral Immunology,
Butantan Institute, Brazil
Ronaldo Zucatelli Mendonc¸a, Laboratory of Viral Immunology, Butantan Institute,
Brazil
A
ˆ
ngela Maria Moraes, Department of Biotechnological Processes, School of Chemical
Engineering, State University of Campinas, Brazil
Ana Maria Moro, Laboratory for Biopharmaceuticals in Animal Cells, Butantan Institute,
Brazil
Ana Cristina Almeida Mu
¨
ller, Carminatti Schulz Propriedade Intelectual, Brazil
Marcia Regina da Silva Pedrini, Federal University of Rio Grande do Norte, Brazil
Maı
´
ra Peixoto Pellegrini, Cell Culture Engineering Laboratory, PEQ/COPPE, Federal
University of Rio de Janeiro, Brazil
Carlos Augusto Pereira, Laboratory of Viral Immunology, Butantan Institute, Brazil
Rosane Aparecida Moniz Piccoli, Institute of Technological Research of the State of Sa
˜
o
Paulo, Brazil
Rodrigo Coelho Ventura Pinto, Cell Culture Engineering Laboratory, PEQ/COPPE,
Federal University of Rio de Janeiro, Brazil
Maria Teresa Alves Rodrigues, Laboratory of Biopharmaceuticals in Animal Cells,
Butantan Institute, Brazil
Jose
´
Maciel Rodrigues Jr, Nanocore Biotecnologia Ltda, Brazil
Gryssell Rodrı
´
guez, Center of Molecular Immunology, Cuba
Sandra Aparecida dos Santos, Nanocore Biotecnologia Ltda, Brazil
Ce
´
lio Lopes Silva, University of Sa
˜
o Paulo, Brazil
Hamilton da Silva Jr, Institute of Biomedical Sciences, Federal University of Rio de
Janeiro, Brazil
Claudio Alberto Torres Suazo, Department of Chemical Engineering, Federal University
of Sa
˜
o Carlos, Brazil
Wirla Maria S. C. Tamashiro, Institute of Biology, State University of Campinas, Brazil
Aldo Tonso, Department of Chemical Engineering, University of Sa
˜
o Paulo, Brazil
Contributors xv
Abbr eviations and Symbols List
Chapter 1
ADA adenosine deaminase deficiency
BEVS baculovirus expression vector system
BHK baby hamster kidney cell line
CHO Chinese hamster ovary cell line
ELISA enzyme linked immuno sorbent assay
mAb monoclonal antibodies
MDCK Madin-Darby canine kidney epithelial cells
MMR measles, mumps, rubella
SCID severe combined immunodeficiency
tPA plasminogen activator
VLP virus-like particle
Chapter 2
ATP adenosine triphosphate
EDTA ethylenediaminetetraacetic acid
DMSO Dimethylsulfoxide
BHK baby hamster kidney cell line
CHO Chinese hamster ovary cell line
HEPES N-(2-hydroxyethyl)piperazine-N9-(2-ethanesulphonic acid)
mAbs monoclonal antibodies
specific cell growth rate
max
maximum specific cell growth rate
MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide)
PEG polyethylene glycol
RNA ribonucleic acid
t cell culture time
t
d
cell doubling time
WHO World Health Organization
X cell concentration
Chapter 3
-gal beta-galactosidase
AAV adeno-associated virus
AP alkaline phosphatase
ARE elements rich in adenosine and uridine nucleotides (AU-rich elements)
BHK baby hamster kidney cells
bp base pairs
BPV bovine papilloma virus
CAT chloramphenicol acetyl transferase
cDNA complementary deoxyribonucleic acid
CHO Chinese hamster ovary cell line
CMV cytomegalovirus
COS African green monkey kidney cells (CV1 cells transformed with a
defective mutant of SV40)
Cre recombinase (cyclization recombination)
CV1 African green monkey kidney cells
DEAE-dextran diethylaminoethyl-dextran
DHFR dihydrofolate reductase
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
DOPE dioleoylphosphatidyl ethanolamine
DOSPA 2,3-dioleoyloxy-N-[2(sperminecarbox-amido)ethyl]-N,N-dimethyl-1-
propane-aminium-trifluoracetate
DOTMA N-[2,3-(dioleyloxy) propyl]-N,N,N-trimethyl ammonium chloride
E. coli Escherichia coli
E erythromycine operon/repressor
EBV Epstein-Barr virus
ECFP enhanced cyan fluorescence protein
ECV extracellular viral particles
EGFP enhanced green fluorescence protein
ELISA enzyme-linked immunosorbent assay
EYFP enhanced yellow fluorescence protein
FACS fluorescence-activated cell sorter
FdG fluorescein di--D-galactopyranoside
FH2 dihydrofolate
FH4 tetrahydrofolate
FK506 human immunophilins
FLP native recombinase isolated from the 2m plasmid from Saccharomyces
cerevisiae
FRAP rapamycin-induced immunophilins
FRT FLP recombination target
G418 geneticin
gDNA genomic DNA
GFP green fluorescence protein
GS glutamine synthetase
GST glutathione S-transferase
HAT hypoxanthine aminopterin thymidine
HBK hybrid cell line generated from the fusion of Burkitt lymphoma and
HEK-293 cells
HEK-293 human embryonic kidney cells
hGH human growth hormone
High-Five TM BTI-TN-5B1-4 (cell line derived from the insect Trichoplusia ni)
hsp70 heat shock protein 70
HSV herpes simplex virus
IPTG isopropyl 1-thio-ß-D-galactopyranoside
IRES internal ribosomal entry site
kb kilobases
Lac lactose operon/repressor
LCR locus control region
LoxP locus of crossover of P1
LUC luciferase isolated from firefly
MCS multiple cloning site
Abbreviations and Symbols List xvii
mRNA messenger RNA
MSX methionine sulfoximine
MTX methotrexate
NSONS0 murine myeloma cell line
ONPG o-nitrophenyl--D-galactoside
ORF open reading frame
OV occluded viral particles
pCMV cytomegalovirus promoter
PCR polymerase chain reaction
PEI polyethyleneimine
PIP pristinamycine
polyA polyadenylation signal
pSV40 early promoter of SV40 virus
RIA radioimmuno assay
RNA ribonucleic acid
RU486 mifepristone
SAR scaffold attached region
SEAP secreted alkaline phosphatase
Sf-21 cell line originated from the ovary of the insect Spodoptera frugiperda
Sf-9 clone derived from the Sf-21 cell line
Sp2/0 mouse myeloma cell line
SV40 simian virus 40
Tet tetracycline operon/repressor
TEV catalytic domain of the nuclear inclusion a (NIa) from the tobacco etch
virus
TK thymidine kinase
Tn-368 cell line originated from the ovary of the insect Trichoplusia ni
Tn7 transposon 7
tPA tissue plasminogen activator
tRNA transfer ribonucleic acid
UTR untranslated regions
X-gal 5-bromo-4-chloro-3-indolyl-ß-D-galactoside
YACs yeast artificial chromosomes
Chapter 4
Acetyl-CoA acetyl-coenzyme A
ADP adenosine diphosphate
Ala alanine
AMP adenosine monophosphate
ATP adenosine triphosphate
BHK baby hamster kidney cell line
CC9C10 hybridoma cell line
CHO Chinese hamster ovary cell line
CHSE Chinook salmon embryo cell line
CMP-NANA cytosine monophosphate-N-acetylneuraminic acid
CoQ coenzyme Q
CoQH
2
dihydroubiquinone
DNA deoxyribonucleic acid
xviii Animal Cell Technology
EPO erythropoietin
FAD flavin adenine dinucleotide
FADH
2
1,5-dihydro- flavin adenine dinucleotide
Fruc 6-P fructose-6-phosphate
GDP guanosine 59-diphosphate
Glc-6-P glucose-6-phosphate
GlcN 6 P glucosamine-6-phosphate
GlcNAc-1-P N-acetylglucosamine-1-phosphate
GlcNAc-6-P N-acetylglucosamine-6-phosphate
Gln glutamine
Gly glycine
GP1 immunoadhesin glycoprotein
GTP guanosine 59-triphosphate
HL-60 human promyelocytic leukaemic cell line
IgG immunoglobulin G
IMDM Iscove9s Modified Dulbecco9s Medium
LLC-PK(1) porcine kidney cell line
mAb monoclonal antibody
ManNAc6P mannose-N-acetyl-6-phosphate
MDCK Madin-Darby canine kidney cell line
MGH-U1 human bladder cancer cell line
MN12 mouse-mouse hybridoma cell line
NAD
þ
nicotinamide adenine dinucleotide
NADH reduced NAD
NADP nicotinamide adenine dinucleotide phosphate
NADPH reduced NADP
NANA N-acetylneuraminic acid
NeuNAc N-acetylneuraminic acid
OK opossum kidney cell line
OMP orotidine monophosphate
P phosphate
P/O ratio molar ratio of ATP synthesis and oxygen consumption
PER.C6 human retina-derived cell line
PP pyrophosphate
PPC pentose phosphate cycle
PQXB1/2 murine B-lymphocyte hybridoma cell line
RNA ribonucleic acid
S I sugar nucleoside transport system I
S II sugar nucleoside transport system II
Sf9 insect cell line (Spodoptera frugiperda)
SP2/0 mouse myeloma cell line
SP2/0-Ag14 mouse myeloma cell line
TCA tricarboxylic acid cycle
TNFR-IgG tumor necrosis factor receptor-IgG
tPA tissue plasminogen activator
UDP uridine diphosphate
UDP uridine diphosphate
UDP-GalNAc UDP-N-acetylgalactosamine
UDP-GlcNAC UDP-N-acetylglucosamine
UDP-NANA uridine diphosphate N-acetylneuraminic acid
Abbreviations and Symbols List xix