New Developments and New Applications
in Animal Cell Technology
New Developments
and New Applications
in Animal Cell Technology
Proceedings of the 15th ESACT Meeting
Edited by
Otto-Wilhelm Merten
Pierre Perrin
and
Bryan Griffiths
KLUWER ACADEMIC PUBLISHERS
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ORGANIZING COMMITTEE:
Otto-Wilhelm Merten (Meeting Secretary)
Wolfgang Noé (Sponsoring, Trading)
Georgia Barlovatz-Meimon
Andres Crespo, Génopoïétic
Jean-Marc Engasser
Fabrice Geoffroy
Jean-Marc Guillaume
Jan Lupker
Annie Marc
Pierre Perrin
v
SPONSORS:
The Organizing Committee acknowledges the financial support to :
Companies:
Bayer
B. Braun Biotech International
Biolnvent Production AB
Boehringer Ingelheim Bioproducts
Boehringer Mannheim
Covance Laboratories Ltd.
Dr. Karl Thomae GmbH
Genzyme Transgenics Corp.
Hoffmann-La Roche Inc.
Hyclone Europe
Intervent International bv
JRH Biosciences
Laboratoires Serono SA
Life Technologies
MA BioServices
Merck KG
Merck Sharp & Dohme
MicroSafe B.V.
Nature-MacMillan Magazines
Novo Nordisk
Nunc A/S
Pharmacia Biotech AB
Promega
Q-One Biotech Ltd.
Sanofi Recherche
Schering AG
SmithKline Beecham Biologicals
Institutions:
City of Tours
European Commission - DGXII - Biology
vi
ESACT Executive Committee, 1996-1997
C. MacDonald Chairperson University of Paisley, Paisley, U.K.
L. Fabry
B. Griffiths Secretary Smith Kline Beecham, Rixensart, B
O.-W. Merten
Treasurer CAMR, Porton, U.K.
M. Carrondo
Meeting Secretary Généthon II, Evry, F
H. Hauser
J. Lehmann IBET/CTQB, Oeiras, P
W. Noé
GBF, Braunschweig, D
University of Bielefeld, Bielefeld, D
Dr. Karl Thomae GmbH, Biberach, D
vii
15th ESACT Meeting, Tours, France
Session chairpersons :
Session 1st Chairperson 2nd Chairperson
E.T. Papoutsakis
1. J. Lehmann W. Scheirer
J.-M. Guillaume
2. G. Barlowatz-Meimon J. Lupker
3. A. Crespo F. Geoffroy
4. B. Griffiths C. MacDonald
J.-M. Engasser
5. L. Fabry J. Bailey
6. H. Hauser
7. M. Carrondo
8. M. Al-Rubeai
Workshop S. Barteling
viii
CONTENTS
Introduction xxiii
HYCLONE Lecture 1
Genetic manipulation of the mammalian genome (Abstract)
R. Jaenisch
ESACT Lecture 5
Engineering glycosylation in animal cells
J.E. Bailey, E. Prati, J. Jean-Mairet, A. Sburlati, P. Umaña
Sessions on : 25
Biosynthesis and post-translational modifications of recombinant proteins,
and Cell physiology and metabolic engineering of animal cells
Gene expression : 27
Enhanced recombinant protein expression in insect cells in the presence of 29
dimethylsulfoxide (DMSO)
L. Ramos, J.F. Kane, A.A. Murnane
Studies of the hepatitis C virus recombinant protein production and its effects 35
on Sf-9 insect cells metabolism
C.M. Charon
Characterization of phosphorylation mutants of the human multidrug transporter 39
(MDR1) expressed in the baculovirus-Sf9 insect cell system
K. Szabo, E. Bakos, E. Welker, A. Varadi, H.R. Goodfellow, C.F. Higgins,
B. Sarkadi
Glutamine synthetase transfected cells may avoid selection by releasing glutamine
P. Bird, E. Bolam, L. Castell, O. Obeid, N. Darton, G. Hale 43
Study of different cell culture conditions for the production of a reshaped 51
Mab in NS0 cells
A.J. Castillo, A. Fernandez, T. Boggiano, P. Pugeaud, I.W. Marison
Increasing monoclonal antibody productivity by semicontinuous substitution of 55
production medium for growth medium
x
Expression of recombinant human insulin in Chinese hamster ovary cells is 59
complicated by intracellular insulin-degrading enzymes 69
S.C.O. Pak, S.M.N. Hunt, M.J. Sleigh, P.P. Gray 77
81
High yield expression of recombinant plasma factors : use of recombinant 85
endoprotease derivatives in vivo and in vitro 93
U. Schlokat, A. Preininger, M. Himmelspach, G. Mohr, B. Fischer, F. Dorner
97
The role of cell cycle in determining levels of gene expression in CHO cells 101
D.R. Lloyd, V. Leelavatcharamas, D.C. Edwards, A.N. Emery, M. Al-Rubeai
Heterogeneity within DHFR-mediated gene amplified population of CHO cells
producing chimeric antibodies
N.S. Kim, S.J. Kim, G.M. Lee
Immunofluorescence detection of recombinant monoclonal antibody as a tool
in the characterization of chinese hamster ovary cell lines
C.A.S. Kinney, R.E. Trala, L.C. Hendricks, T.D. Hill
Electrolyzed reduced water which can scavenge active oxygen species supresses
cell growth and regulates gene expression of animal cells
S. Shirahata, S. Kabayama, K. Kusumoto, M. Gotoh, K. Teruya, K. Otsubo,
T. S. Morisawa, H. Hayashi, K. Katakura
Differential gene expression of cytochrome P450 in immortalised hepatocyte
cell lines
H.T. Kelly, K. Anderson, E. Hill, H. Grant, C. MacDonald
Post-transcriptional control of recombinant gene expression : mRNA retargeting
and regulation of expression
K.A. Partridge, A. Johannessen, A. Tauler, I.F. Pryme, J.E. Hesketh
Transient transfection in mammalian cells. A basic study for an efficient and cost- 105
effective scale-up process
E.-J. Schlaeger, L.Y. Legendre, A. Trzeciak, E.A. Kitas, K. Christensen,
U. Deuschle, A. Supersaxo
Large scale transient gene expression in mammalian cells 113
E.-J. Schlaeger, K.Christensen, G. Schmid, N. Schaub, B. Wipf, A. Weiss
xi
Synergistic enhancement of transient expression by dioleoyl-melittin (DOM) 117
and polyethylenimine (PEI) in mammalian cells in suspension culture
E.-J. Schlaeger, J.Y. Legendre, A. Trzeciak, E.A. Kitas, K. Christensen,
U. Deuschle, A. Supersaxo
Transient expression of a soluble and secreted form of heterodimeric T-cell 121
receptor in HEK-293 125
M. Jordan, C.L. Blanchard, I. Bernasconi, I. Luescher, F.M. Wurm
Measurable parameters of cells and precipitate predict transfectability with
calcium phosphate
M. Jordan, F. Wurm
Glycosylation: 129
131
Influence of Na-butyrate on the production and sialylation of human
interferon-g by a2,6-sialyltransferase engineered CHO-cells
D. Lamotte, L. Monaco, N. Jenkins, A. Marc
Elevated inhibits the polysialylation of the neural cell adhesion molecule
in CHO MT2-1-8 cell cultures 135
J.A. Zanghi, T.P. Mendoza, R.H. Knop, W.M. Miller
Influence of cultivation conditions on glycosylation pattern - a fed-batch and 141
continuous culture study 149
N.A. Schill, M.Z. Rosenberg, R.L. Dabora 153
Effects of different production systems on glycosylation pattern of murine
monoclonal IgA
F. Schweikart, E. Lüllau, R. Jones, G.J. Hughes
Identification of altered glycosylation as the major difference between
intracellulary accumulated and secreted b-trace protein produced in baculovirus-
infected insect cells
G. Rodriguez, H.S. Conradt, V. Jäger
xii
How ammonium dominates the metabolism of in vitro cultivated mammalian 157
cells
A. Çayli, M. Wirth, R. Wagner
Study of human recombinant GM-CSF produced in different host systems using 163
monoclonal antibodies
M. Etcheverrigaray, M. Oggero, M. Bollati, R. Kratje
Metabolic engineering : 166
Modification of hybridoma cells metabolism 167
J.J. Cairó, C. Paredes, F. Gòdia, E. Prats, F. Azorín, Ll. Cornudella
Cloning and expression of a cytosolic sialidase from CHO cells in a glutathione 175
S-transferase (GST)-encoding expression vector
M. Burg, J. Müthing
Construction of a novel CHO cell line coexpressing human glycosyltransferases 181
and fusion PSGL-1 - immunoglobulin G
B. Vonach, B. Hess, C. Leist
Production of defined glycosylation variants of secreted human of secreted human 185
glycoprotein therapeutics by coexpression with human recombinant
glycosyltransferases in BHK-21 cells
P. Schlenke, E. Grabenhorst, J. Costa, M. Nimtz, H.S. Conradt
Antisense RNA for the elimination of NeuGc residues from recombinant 191
glycoproteins
A. Gregoire, A. Visvikis, A. Marc, J.-L. Goergen
Cell proliferation, proliferation control, apoptosis: 195
Intracellular fatty acid composition affects cell yield, energy metabolism and 197
cell damage in agitated cultures
M. Butler, N. Huzel, N. Barnabé, L. Bajno, T. Gray
Lipid requirements of a recombinant Chinese hamster ovary cell line (CHO) 205
J.T. McGrew, C.L. Richards, P. Smidt, B. Dell, V. Price
Sustained expression in proliferation controled BHK-21 cells xiii
P.P. Müller, S. Kirchhoff, H. Hauser 209
215
Cell growth inhibition by the IRF-1 system 219
A.V. Carvalhal, J.L. Moreira, P. Müller, H. Hauser, M.J.T. Carrondo
223
Correlation between BHK cell specific productivity and metabolism 227
H.J. Cruz, J.L. Moreira, E.M. Dias, C.M. Peixoto, A.S. Ferreira, 231
235
M.J.T. Carrondo 243
Effect of growth arrest in BHK metabolism: on line monitoring by 13C and 31P 247
255
NMR spectroscopy 259
P.M. Alves, A.V. Carvalhal, J.L. Moreira, H. Santos, M.J.T. Carrondo
Cell survival in CHO cell cultures grown in a defined protein free medium
N. Chatzisavido, C. Fenge, L. Häggström
Enhanced apoptosis in insect cells cultivated in simulated microgravity
N. Cowger, K. O’Connor
Modulation of apoptosis by bcl-2 expression following amino acid deprivation
and in high cell density perfusion cultures
R.P. Singh, D. Fassnacht, A. Perani, N.H. Simpson, C. Goldenzon, R. Pörtner,
M. Al-Rubeai
Effect of bcl-2 expression on hybridoma cell growth during stressful conditions
D. Fassnacht, S. Rössing, , M. Al-Rubeai, R. Pörtner
Inhibition of c-jun expression in F-MEL cells causes cell cycle arrest and
prevention of apoptosis
Y.H. Kim, T. Iida, E.V. Prochownik, E. Suzuki
Hippocampal cells in culture as a model to study neuronal apoptosis
I. Figiel, J. Jaworski, L. Kaczmarek
Development of carboxy SNARF-1-AM and annexin V assays for the
determination of apoptosis in heterogeneous cultures
A. Ishaque, M. Al-Rubeai
xiv
Session on : 263
Integrated bioprocessing in animal cell technology
Physical environment, cells and products : 265
Diauxic cell behavior enables detoxification of CHO cell culture medium during 267
fed batch cultivation
H. Lübben, G. Kretzmer
Effects of polysaccharide derived from tea on growth of human cell lines in 273
serum free culture
H. Kawahara, M. Maeda-Yamamoto, K. Osada, K. Tsuji
Metabolic demands of BTI-TN-5B1-4 (High Five™) insect cells during growth 277
and after infection with baculovirus
E. Chico, V. Jäger 281
Effects of ammonium and lactate during continuous hybridoma fermentations 285
in a fluidized-bed bioreactor 293
H. Heine, M. Spies, M. Biselli, C. Wandrey 303
307
Serum concentration and pH affect the CD13 receptor content of HL60 cells 309
cultured in stirred bioreactors
C.L. McDowell, E.T. Papoutsakis 317
Four regulation-friendly serum-free media for mammalian and insect cells
T.W. Irish, S.E. Lenk, L.A. Bugner, K.J. Etchberger
Proteolytic activities in the baculovirus-insect cell expression system
G. Schmid, A. Bischoff
Monitoring and control:
Development of proteinase assays for improved CHO cell cultures
C. Tans, C. vander Maelen, S. Wattiaux-de Coninck, M.-M. Gonze, L. Fabry
Digital image analysis: quantitative evaluation of colored microscopic images
of animal cells
K. Falkner, E.D. Gilles
xv
The viable cell monitor: a dielectric spectroscope for growth and metabolic 321
studies of animal cells on macroporous beads
Y. Guan, R.B. Kemp
Measurements of changes in cell size distribution to monitor baculovirus 329
infection of insect cells
E. Chico, V. Jäger
Dead cell estimation - a comparison of different methods 333
A. Falkenhain, Th. Lorenz, U. Behrendt, J. Lehmann
Fed-batch culture development based on biomass monitoring 337
E. de Buyl, A. Maxwell, L. Fabry
On-line monitoring of protein and substrate/product concentration in mammalian 343
cell cultivation
H. Lübben, J. Hagedorn, T. Scheper
Process control and on-line feeding strategies for fed-batch and dialysis cultures 347
of hybridoma cells
J.O. Schwabe, R. Pörtner
Metabolic network analysis of a hybridoma cell line using mass balances 351
and labelled glucose and glutamine
P.-A. Ruffieux, I.W. Marison, U. von Stockar
Dynamic medium optimization by on-line heat flux measurement and a 355
stoichiometric model in mammalian cell culture
Y. Guan, R.B. Kemp
Modeling of glycoprotein production by chinese hamster ovary cells for process 359
monitoring and control
J. Stelling, R.K. Biener, J. Haas, G. Oswald, D. Schuller, W. Noé, E.D. Gilles
The temperature effect in mammalian cell culture. An Arrhenius interpretation 363
G. Kretzmer, T. Buch, K. Konstantinov, D. Naveh
Integrated processes and scale-up: 367
Dielectrophoretic forces can be exploited to increase the efficiency of animal cell 369
perfusion cultures
N. Kalogerakis, A. Docoslis, L.A. Behie
xvi 377
Use of a new microcarrier with two-dimensional geometry for the culture of 381
anchorage-dependent cells in sonoperfused, continuously stirred tank reactors 385
C. Gatot, F. Trampler, M.-P. Wanderpepen, J. Harfield, A. Oudshoorn, 389
A. Johansson, V. Nielsen, A.O.A. Miller 395
399
Evaluation of porous microcarriers in fluidized bed reactor for protein 409
production by HEK 293 cells 417
M.A. Valle, J. Kaufman, W.E. Bentley, J. Shiloach
421
Fluidized bed technology: influence of fluidization velocity on nutrient 429
consumption and product expression
G. Blüml, M. Reiter, Th. Gaida, N. Zach, A. Assadian, C. Schmatz, H. Katinger
High density and scale-up cultivation of recombinant CHO cell line and
hybridomas with porous microcarrier cytopore
C. Xiao, Z. Huang, W. Li, X. Hu, W. Qu, L. Gao, G. Liu
Cell-settler perfusion system for the production and glycosylation of human
interferon-γ by clumped cells
D. Lamotte, J. Straczek, A. Marc
Disposable bioreactor for cell culture using wave-induced agitation
V. Singh
Use of miniPERM system for an efficient production of mouse monoclonal
antibodies
M.L. Nolli, R. Rossi, A. Soffientini, D. Zanette, C. Quarta
Production of anti-EGF receptor hMAb in hollow fiber bioreactors for in vivo
diagnosis
M.A. Arias, A. Valdés, D. Curbelo, O.M. Morejón, I. Caballero, J. Villán,
J.A. Gómez, A. Fernandéz, J. Rodriguez, A. Morales, T. Boggiano, A. Castillo,
L. Bouzó, C. Hermida, G. García, P. Vitón, N. Pérez, T. Rodríguez
Optimisation of the production of VLP’s in 2 Lt stirred bioreactors
P.E. Cruz, A. Cunha, C.C. Peixoto, J. Clemente, J.L. Moreira, M.J.T. Carrondo
Direct capture of monoclonal antibodies with a new high capacity streamline
SP XL ion exchanger
C. Priesner, N. Ameskamp, D. Lütkemeyer, J. Lehmann
The effect of different purification schemes on the activity of a monoclonal xvii
antibody 433
D.J. Black, J.P. Barford, C. Harbour, A. Fletcher 437
441
Interfacing of protein product recovery with an insect cell baculovirus 445
production system 449
I.A. Carmichael, M. Al-Rubeai, A. Lyddiatt
457
Preparation of plasmid DNA using displacement chromatography 463
R. Freitag, S. Vogt 467
469
Novel methods for large-scale preparation of nutrient media and buffered salt 473
solutions 481
D.W. Jayme
Large scale application of the Semliki Forest Virus system: 5-HT3 receptor
production
H.D. Blasey, B. Brethon, R. Hovius, K. Lundström, L. Rey, H. Vogel,
A.-P. Tairi, A.R. Bernard
From applied research to industrial application: The success story of monitoring
intracellular ribonucleotide pools
S.I. Grammatikos, K. Tobien, W. Noé, R.G. Werner
New technology of production recombinant human erythropoietin for peroral
administration
T.D. Kolokoltsova, N.E. Kostina, E.A. Nechaeva, N.D. Yurchenko,
O.V. Shumakova, A.B. Rizikov, N.A. Varaksin, T.G. Ryabicheva
General safety aspects:
Significance of multiple testing on murine leukemia virus of mouse hybridomas
E.J.M. Al, T. Jorritsma, A. Blok, H.M.G. Sillekens, P.C. van Mourik
Murine retrovirus detection using Mus dunni cells and S+L- cells
P. Seechurn, B. Mortimer, P. Newton, C. Martin
The removal of model viruses during the purification of human albumin using
chromatographic procedures
R. Cameron, C. Harbour, Y. Cossart, J.P. Barford
xviii 485
491
Inactivation of hepatitis A virus during the production of Nordimmun 493
D.W. Birch, P. Kaersgaard, C. Martin 503
509
Session on: 515
Viral vector production for gene therapy 523
531
Adeno-associated viral vectors: Principles and in vivo use 541
O. Danos
545
Novel retroviral packaging cell lines: Improved vector production for efficient 549
hematopoietic progenitor cell transduction 551
S. Forestall, R. Rigg, J. Dando, J. Chen, B. Joyce, R. Tushinski, C. Reading,
E. Böhnlein
Scale-up and otpimisation issues in gene therapy. Viral vector production
J.E. Boyd, L. Borland, C.E.Fisher
Comparison of manufacturing techniques for adenovirus production
J.M. Ostrove, P. Iyer, J. Marshall, D. Vacante
Safety considerations in the development of new retroviral and adenoviral
vectors for gene therapy
D. Morgan, I. Forgie, J. Ostrove, M.H. Wisher
Gene therapy for neuromuscular disorders : Macrophages as shuttles for
widespread targeting
E. Parrish, E. Peltékian, L. Garcia
Gene transfer into dog myoblasts
S. Braun, C. Thioudellet, C. Escriou, M.-C. Claudepierre, F. Längle-Rouault,
E. Jacobs, R. Bischoff, D. Elmlinger, H . Homann, Y. Poitevin, M. Lusky,
M. Mehtali, F. Perraud, A. Pavirani
Biodistribution analysis of a gene therapy vector using the polymerase chain
reaction (PCR) technique
J. Proffitt, M. Leibbrandt, K. Jarvis, C. Martin
Session on:
Developments for immunologicals and vaccines
The production of influenza virus by immobilised MDCK cells
D. Looby, J. Tree, A. Talukder, K. Hayes, H. House, G. Stacey
Serum-free grown MDCK cells: An alternative for influenza vaccine virus xix
production 555
N. Kessler, G. Thomas, L. Gerentes, M. Aymard 561
569
Evaluation of the new medium (MDSS2N), free of serum and animal proteins, 573
for the production of biologicals
H. Kallel, P. Perrin, O.-W. Merten 577
581
Production of high titre disabled infectious single cycle (DISC) HSV-2 from a 583
microcarrier culture 593
T.A. Zecchini, R.J. Smith 597
601
New form of the live measles vaccine for oral administration 607
E.A. Nechaeva, E.A. Kashentseva, A.P. Agafonov, N.A. Varaksin,
T.G. Ryabicheva, A.P. Konstantinov, V.N. Bondarenko, T.D. Kolokoltsova,
I.V. Kits, T.Yu. Sen’Kina, N.V. Zhilina
Study of Leningrad-16 vaccine strain of measles virus reproduction in cell
cultures perspective for biotechnology
E.A. Nechaeva, T.N. Getmanova, T.Y. Sen’Kina, N.D. Yurchenko
Recent advances with new vaccine adjuvants from preclinical to clinical
development (Abstract)
T. Voss
Vaccination with recombinant suicidal DNA/RNA
P. Berglund, M. Fleeton, C. Smerdou, I. Tubulekas, B.J. Sheahan, G.J. Atkins,
P. Liljeström
Growth of goat endothelial cells for the production of a veterinary vaccine
P.M. Miranda, J.L. Moreira, M.J.T. Carrondo
Expression in insect cells of the major parasite antigen associated with human
resistance to Schistosomiasis
L.Argiro, C. Doerig, S. Liabeuf, A. Bourgois, J.L. Romette
Modulation of CD4 expression on helper T lymphocytes and U937 cells by
ganglioside GM3 and its derivatives
D. Heitmann, P. Budde, J. Frey, J. Lehmann, J. Müthing
Human serum in leukocyte cultures producing human interferon alpha
P. Mattana, L. Scapol, S. Silvestri, G.C. Viscomi
xx
The new type of immunomodulator 613
M.V. Mezentseva, V.A. Mozgovoi, L. Yu. Mozgovaya, R. Ya. Podchernyaeva 617
625
In vitro immunization of human peripheral blood lymphocytes with cholera 627
toxin B subunit 635
A. Ichikawa, Y. Katakura, T. Kawahara, S. Hashizume, S. Shirahata 637
645
Session on: 657
New technologies for health care products 661
665
Analysis of cell growth in a fixed bed bioreactor using magnetic resonance 669
spectroscopy and imaging
P.E. Thelwall, M.L. Anthony, D. Fassnacht, R. Pörtner, K.M. Brindle
Expansion of human hematopoietic progenitor cells in a fixed bed bioreactor
(Abstract)
P. Meissner, P. Werner, B. Schröder, C. Herfurth, C. Wandrey, M. Biselli
A novel assay to determine and quantify the regulation of cell motility and
migration demonstrated on hematopoietic cells
D. Möbest, S. Ries, R. Mertelsmann, R. Henschler
Immortalization of differentiated hepatocytes
G.S. Jennings, M. Strauss
Fixed-bed reactors for animal cell cultivation: An approach to artificial organs
R. Pörtner, S. Rössing, J. Stange, D. Fassnacht
High density perfusion culture of primary rat hepatocytes for potential use as a
bioartificial liver device
K. Bratch, A.J. Strain, M. Al-Rubeai
Metabolic competence and hormonal regulation of primary porcine hepatocytes
in a 3-D sandwich configuration (Abstract)
A. Bader, D. Rocker, A. Acigköz, S. Schwintek, J. von Schweder, M. Maringka,
V. Armstrong, R. Wagner, G. Steinhoff, A. Haverich
Novel mini-bioreactors for islet cell culture
A. Handa-Corrigan, I.C. Green, J. Mabley, S. Hayavi, G.N. Kass, R.H. Hinton,
L.M. Morgan, J. Wright
xxi
Cultivation of skin cells suitable for recovery of burn wounds 673
T.D. Kolokoltsova, N.D. Yurchenko, N.G. Kolosov, O.V. Shumakova,
E.A. Nechaeva
Tissue therapy for treatment of primary myodystrophies 677
T.B. Krokhina, G.B. Raevskaja, S.S. Shishkin
Possibility of application of thyroid organ culture for the treatment of persistent 681
hypothyroidism
I.P. Pasteur, N.D. Tronko, E.N. Gorban, V.I. Kravchenko
ESACT Lecture 685
Human cells as therapeutic agents (Abstract) 687
H . Green 689
705
Session on: 713
Use of animal cells for in vitro testing 717
Embryonic stem cell differentiation models: Cardio-vascular, myogenic and
neurogenic development in vitro
A.M. Wobus, K. Guan, J. Rohwedel, C. Strübing, M. Drab
Responses of human lung epithelial cells (A549) to pathogenic infection by
Mycoplasma pneumoniae
J. Goodman, K. Morley, P. Packer, T. Battle
Polar lipid profiling of Mycoplasma pneumoniae-infected human lung epithelial
cells
J. Goodman, R. Wait, T. Battle
Comparison of three dimensional (3-D) rat hepatocyte cultures in simulated
microgravity conditions
T. Maguire, H.J. Moulsdale, G. Stacey, T. Battle
High density culture of the human hepatoma cell line HepG2: Long-term culture 721
for in vitro toxicology 725
A. Handa-Corrigan, R.M. Traynor, I. Adamopoulos, J. Salway
Application of primary cultures of rat fetal neurons to the study of neurotrophic
action of peptides
O.V. Dolotov, I.A. Grivennikov
xxii
Development and validation of an image analysis system for single cell 729
characterization in cell monolayers
D. Kaiser, M.A. Freyberg, G. von Wichert, P. Marenbach, H. Tolle, P. Friedl
Development of an optically accessible perfusion chamber for in situ assays and 733
for long-term cultivation of mammalian cells
M.A. Freyberg, P. Friedl
Analysis of mitogenic activity of proteins after separation by gel electrophoresis 737
O. Hohenwarter, G. Marzban, E. Jisa, H. Katinger
Workshop on: 741
The use of animal cells versus the use of transgenic animals for the 743
production of recombinant proteins
Secretion of fusion proteins into milk by transgenic mouse mammary epithelium
(Abstract)
D. Pollok, L. Chen, H. Liem, B. Wilburn, J. Williams, M. Harrington,
Y. Echelard, H. Meade
The production of proteins in the milk of transgenic livestock: A comparison of 745
microinjection and nuclear transfer 751
I. Garner
757
Creation of mice expressing human antibody by introduction of a human 765
chromosome
H. Yoshida, K. Tomizuka, H. Uejima, H. Kugoh, K. Satoh, A. Ohguma,
M. Hayasaka, K. Hanaoka, M. Oshimura, I. Ishida
Transgenic technology - A challenge for mammalian cell culture production
systems
R.G. Werner
General Workshop-Discussion
List of trade-fair-sponsors 773
Index 775
INTRODUCTION
New Developments and New Applications in Animal Cell Technology
The 15th Meeting of the European Society for Animal Cell Technology (ESACT),
entitled « Animal Cell Technology. New Developments - New Applications », was
organized in Tours/France at the beginning of September 1997. The objective was to
give an overview of the actual and future developments and on new applications in the
classical fields covered by ESACT as well as in adjacent areas. Products, like vaccines
and recombinant proteins are standard products of aanimal cell technology, and have been
covered by ESACT-Meetings for many years. The actual trend in the use of animal cell
technology is going towards new domains which have not been covered previously or
sufficiently by ESACT, and of which ESACT should be more aware. This is because it
can provide a lot of technological expertise: for instance, the development, production,
and use of viral vectors for use in gene therapy is comparatively new and has a bright
future, or the use of cell culture technology for the development of artificial organs
which is steadily becoming more important, as specially as established technologies for
entrapment and immobilisation of animal cells are of utmost importance in this field.
ESACT is a Society of applied scientists and engineers. It is therefore very important for
members to get a deeper insight into the actual developments in fundamental research,
because in the near or further future they have to deal with the results of this research by,
for instance, developing production, cultivation, or purification methods. In order to
fulfil these requirements (getting a view on these subjects and also getting overviews on
matters of general interest for applied biotechnologists), several keynote lectures were
organized during the 15th ESACT-Meeting. It was possible to get five keynote speakers
and the speaker of the opening session (F. Horaud, Inst. Pasteur, Paris/F) all of whom
participating actively at this meeting. These speakers gave excellent talks on diverse
subjects which included DNA methylation and its role in the genetic manipulation of the
mammalian genome (R. Jaenisch, Whitehead Inst., Cambridge, MA); the use of human
cells as therapeutic agents (H. Green, Harvard Medical School, Boston, MA), the
engineering of glycosylation patterns in mammalian cells (J. Bailey, ETH Zürich, CH),
and how genomics, target identification and structure based design could provide a
complementary rather than competitive approach to animal cells for human therapy (T.
Blundell, University of Cambridge, U.K.). Finally a more global overview on the
reshaping of medicine through molecular biology was given by T. Bartfai (Hoffmann La
Roche, Basel/CH).
During the 15th ESACT-Meeting eight scientific sessions and one workshop were
organized :
1. New technologies for health care products,
2. Use of animal cells for in vitro testing.
xxiii
xxiv
3. Viral vector production for gene therapy,
4. Biosynthesis and post-translational modifications of recombinant proteins,
5. Developments for immunologicals and vaccines,
6. New cell lines: from transformed to differentiated cells/cell functions,
7. Integrated bioprocessing in animal cell technology,
8. Cell physiology and metabolic engineering of animal cells,
and a workshop on: The use of animal cells versus the use of transgenic animals for the
production of recombinant proteins.
The scientific presentation of these Proceedings is in a slightly different fromate to that
of the 15th ESACT-Meeting, nevertheless they reflect very closely the scientific contents
of the meeting. There are two main reasons for the modified presentation: 1.
Scientifically, it was preferable to merge sessions 4 and 8 in order to get a more global
presentation of the biosynthesis of recombinant proteins, cell physiology and metabolism
(including metabolic engineering). 2. There is no chapter with respect to session 6,
because only two manuscripts have been collected which were finally attributed to
sessions 1 (paper by Jennings & Strauss) or 2 (paper by Wobus et al.).
In order to get a better structure of this volume, the chapters corresponding to each
session are introduced by a short overview on the contents.
It is obvious that these Proceedings will be most valuable to those who are actively
involved in the field of animal cell technology. The chapters on Biosynthesis and post-
translational modifications of recombinant proteins and Cell physiology and metabolic
engineering of animal cells and on Integrated bioprocessing in animal cell
technology », representing the key-domaines of ESACT, count for about 60% of all
presentations. Classical fields, like reactor engineering, development and application of
new devices for high cell density rector systems, monitoring, and optimisation are still of
large interest. However, the emphasis in optimisation of the product has changed from
reactor engineering to cell engineering, for instance, for modifying glycosylation patterns
or for reducing apoptosis in reactor cultures (= prolongation of the life time of the active
biomass) becomes of growing interest.
There are many highlights of general interest scattered through the other sessions; among
others to be mentioned, several papers in the session on Viral vector production for
gene therapy on the biology and the potential use of adeno associated virus (AAV) in
gene therapy, the development of new packaging cell lines for the production of viral
vectors, and the application of gene therapy for the treatment of muscular diseases;
papers presenting new approaches in vaccine development (e.g. new adjuvants and new
viral vectors) in the session on (« Developments for immunologicals and vaccines ».)
A topic in an adjacent field of ESACT but of general interest, is the use of high cell
density (tissue-like) for the development of artificial organs. Originally, techniques
developed for hollow-fiber and general immobilisation systems were the base for
developments in the field of artificial organs. Today, general animal cell technology
profits by developments in this field, like techniques for determining the distribution of
biomass in the extracapillary space of high density hollow fiber systems.
xxv
As already mentioned, due to the keen interest a workshop on The use of animal cells
versus the use of transgenic animals for the production of recombinant proteins was
organized. The production of recombinant proteins in transgenic animals might become a
very important concurrence for animal cell technology. The aim of this workshop was to
give a comparison between both technologies, to present the advantages and
disadvantages of both technologies, followed by a general discussion. As a conclusion, it
seems that production in animals is more economical, however, the system is less defined
and this may hamper the registration of the product. Furthermore, the production of
pharmaceuticals such as insulin could leak into the blood and consequently impair the
health of the animal, which is, of course, never a problem in animal cell culture.
In conclusion, as for previous ESACT-Meetings, the whole animal cell production
process was considered - from the initial genetic studies of the cells through the stages of
production to the regulatory issues surrounding product release. The holistic approach to
animal cell technology which is the base of each ESACT-Meeting is the main reason for
the continual increasing interest in these meetings.
O.-W. Merten, P. Perrin, B. Griffiths
Editors
Hyclone Lecture
GENETIC MANIPULATION OF THE MAMMALIAN GENOME
R. JAENISCH
Whitehead Institute for Biomedical Research and Department of
Biology
M.I.T.
Cambridge, MA 02142, USA
Abstract: Over the last decade methods have been developed that allow us to precisely
alter genetically the germ line of mice, permitting the generation of mouse models for
human diseases. In this talk I will first review the technology of gene targeting. I will
then illustrate the application of this technology for studying the role of DNA
methylation in embryonic development and genomic imprinting. DNA methylation is
the most important epigenetic change because DNA methylation is an epigenetic
modification : that is, it is altered during normal development as well as during various
diseases such as cancer. The implications of DNA methylation for biomedical research
will be discussed.
Discussion
Danos: The other drugs that have been used for re-activation of gamma
globulins, like hydroxyurea (HU), reduce methylation. Can they be
used in a cancer setting as well?
Jaenisch: I believe HU gets cells into cycling and that is how you may get
different types of cells; maybe foetal cells which express gamma
globulin genes. I am not sure what the mechanism is, but there is
no real evidence that HU leads to direct de-methylation of those
genes. I should add that HU only works for sickle cell anaemias
and not for telosaemias.
Faff: Is there an enzymatic assay for methyl transferase?
Jaenisch: Yes, there are very good in vitro assays. You take the purified
enzyme, or an extract, label the methyl group and then measure the
transfer of methyl to a substrate.
Singhvi: Are you working with any pharmaceutical company on the
development of drugs for de-methylation?
Jaenisch: Yes, I am very keen on that.
Sasaki: Would it be possible to summarise what you know about the
enzyme responsible for de novo synthesis?
1
O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 1-3.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
Bernard: 3
Jaenisch: believe that they lose the X chromosome gene expression and this
would be sufficient to explain the death but is probably not the
Aunins: whole answer. We do not really know what the role of methylation
Jaenisch: is in development. We know it is important for gene expression
and we have evidence for its importance in gene stability. It might
be important for repair processes. In prokaryotes, methylation is
crucial to mark the parental from daughter strain so you know
whether replication errors occur. There is no evidence for this role
in mammals but it is probable. Methyl transferase is not an
oncogene in the conventional usage but I think it is an oncogenic
determinate.
Can you make a partially inactive mutant of methyl transferase in
vitro?
We have partially inactivated mutants in mice, so we can adjust the
enzyme levels genetically down to any level (2%, 5%, 10%). In
vitro the truncated enzyme has activity. In answer to your
question, I am sure you can but I am not sure why one would want
to do it.
Can you speculate on whether methylation has a role in productivity
of recombinant cell lines in vitro?
The old evidence is that a primary cell, which produces all sorts of
proteins when put in tissue culture, very soon shows de novo
methylation of CPG islands of genes which are tissue specific. So
many genes turn off which are not needed in tissue culture. You
turn off genes which inhibit cell growth, eg myo D is never
methylated in vivo, only in culture. In tissue culture I think de novo
methylation is a growth promoting process.
ENGINEERING GLYCOSYLATION IN ANIMAL CELLS
J. E. Bailey, E. Prati, J. Jean-Mairet, A. Sburlati, P. Umaña
Institute of Biotechnology, ETH Zürich, CH-8093 Zürich, Switzerland
1. Abstract
Glycosylation can have significant effects on activity, pharmacokinetics, targeting,
immunogenicity, and stability of a glycoprotein. Therefore, glycosylation engineering
of animal cells which express cloned glycoprotein products is an enabling technology
for generating molecular and functional diversity of these products. This review
considers potential targets in modifying oligosaccharides on particular glycoprotein
pharmaceuticals, strategies available to guide genetic design of a modified
oligosaccharide biosynthesis pathway which will achieve the desired end-product, the
current challenges and limitations, technologically and scientifically, in achieving
industrially significant results, and progress being made to address these challenges.
2. Introduction
Glycoproteins mediate many essential functions in human beings, other eucaryotic
organisms, and some procaryotes, including catalysis, signalling, cell-cell
communication, and molecular recognition and association. The function of
glycoproteins can be profoundly influenced by their oligosaccharide component(s).
Unfortunately, few simple, general principles are available which unify understanding of
oligosaccharide structure - glycoprotein relationships (here subsequently referred to more
briefly as “structure-function relationships”). Accordingly, the scope of scientific and
technological investigation in this field is vast and cannot be comprehensively
summarized here.
There are two main results from prior work which are critical in this discussion.
1. The oligosaccharide structures which are assembled starting from particular initiation
sites on the polypeptide backbone are functions of the polypeptide amino acid sequence,
the host cell in which the glycoprotein is synthesized, and that cell’s environment.
2. With polypeptide sequence, host cell, and environment fixed, the glycoprotein
molecules which are synthesized differ with respect to their oligosaccharide structures.
5
O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 5-23.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
6
These related yet distinct molecular species are called “glycoforms.” Thus, reference to a
particular glycoprotein is, usually, shorthand for a population of glycoforms.
A definition of “glycosylation engineering” is needed here in order to focus this
presentation (Further, a definition which reasonably delimits the field under discussion is
essential to give that definition any functional utility). Here glycosylation engineering
is considered as a subset of metabolic engineering, defined as “the improvement of
cellular activitites by manipulation of enzymatic, transport, and regulatory functions of
the cell with the use of recombinant DNA technology” [1]. That review included this
perspective on glycosylation engineering, presenting several examples in which genetic
engineering of the oligosaccharide synthesis pathway achieved altered glycosylation of
glycoproteins expressed in mammalian cell culture. A later review took a broader view
of glycosylation engineering, including effects of mutations and cell environment on
glycosylation [2]. Although environmental effects belong to the field of bioprocess
engineering in the current lexicon of our field, some highlights from such bioprocess
research are also included here.
Another specification is required here: what kinds of glycoproteins will be discussed, and
in what context? Here we focus on cloned, heterologous, secreted glycoproteins produced
in animal cell culture, insect cell culture, and, to a lesser degree, in transgenic animals.
These glycoproteins, some of which are listed in Table 1, are the backbone of the
biotechnology industry which has emerged in the last two decades and which has
redefined the scope and significance of animal cell technology in the same period. Of
course glycosylation of viruses has major technological relevance, and glycosylation
must be critically important, in ways still to be elucidated, in tissue engineering, but
these topics must await a future presentation. Further information on the science of
glycobiology, as well as introductions to basic concepts in the field, may be found in a
number of excellent recent reviews [3,4,5,6,7],
The secreted proteins of interest here are used in many applications as injectable
therapeutic agents. This then delimits the set of glycoprotein functional characteristics
of greatest importance. Some of the available information on these is summarized next,
7
concentrating specifically on particular glycoproteins which are current or pending
products of the biotechnology industry.
3. Oligosaccharide Structure-Glycoprotein Function Relationships
The advent of biotechnology enabled synthesis of human polypeptides in heterologous
cells. This new combination of polypeptide and host gave rise to glycoproteins which
had not previously existed, but which were under development as potential therapeutic
agents. This motivated extensive development of more sophisticated methods for
characterization of glycosylation, and also research to explore the implications of
glycosylation on the therapeutic properties of glycoproteins.
As summarized in Table 2, this research has revealed that, in particular cases,
glycosylation can influence the physical properties, immunological interactions,
biological activity, and pharmacokinetics of particular glycoproteins. Unfortunately, it
can be very misleading to generalize from an example for a specific glycoprotein to
other glycoproteins. Oligosaccharides have different roles on different glycoproteins with
different functions. In fact, one group of biochemists believes that oligosaccharides
usually have only generalized, physicochemical effects, while another emphasizes
evidence of specific biological functions of oligosaccharides. Evidence for both
viewpoints exists, prompting the cogent title of one of the foremost reviews of the
subject; “Biological roles of oligosaccharides: all theories are correct” [4].
8
There are a few points which admit generalization: absence of sialic (neuraminic) acid on
the termini of complex oligosaccharides results in more rapid clearance, as does the
presence of high mannose oligosaccharides. These implications of glycosylation have
motivated choice of host cell lines for production of biologicals (to avoid high mannose
structures), and also great emphasis on sialylation in process and product development
and in product characterization.
4. Why do Glycosylation Engineering?
Glycosylation engineering offers a way to generate glycoproteins which are novel in the
sense that they carry different oligosaccharides compared to previously available
material. In view of the sensitivity of function of many early products of the
biotechnology industry to glycosylation, glycosylation engineering is an obvious
strategy for generating “second generation” products. A prominent example is “novel
erythropoiesis-stimulating protein” (NESP), moving very rapidly now through clinical
trials. This product is a preparation of one or several high-activity glycoforms of EPO
[I8]. While such a material can be produced by fractionation of a first-generation
product, glycosylation engineering offers an alternative route to obtain novel
glycoforms, including those not available from the original production cell line.
Glycosylation engineering provides a means to optimize a new product with respect to
function, and also with respect to business position. Novel glycoforms are a possible
basis for patent protection of a product. For example, U.S. Patent 5,547,933 [19]
claims “A non-naturally occurring EPO glycoprotein product... (with) a higher
molecular weight than human urinary EPO..”. Here no glycosylation engineering was
involved - glycosylation different from that of human urinary EPO arises simply
because the EPO referred to is made in recombinant CHO cells. Nonetheless, by choice
of a different host, modification of the host by glycosylation engineering, or by
innovative purification strategies, clear opportunities exist for creating new materials
which can be patented.
Glycosylation engineering may also be employed to facilitate purification (e.g., by
installing a residue or linkage recognized by a particular lectin), to simplify
crystallization (in the limiting case, by modifying the amino acid sequence so that it is
not glycosylated), or to reduce glycoform heterogeneity in a glycoprotein preparation
[2,20].
With these practical benefits as motivations, we next review the factors which influence
glycosylation of a polypeptide, which leads us to questions of how glycosylation can
differ among glycoforms, or among different glycosylated polypeptides. Each of these
types of characteristics, and these different ways of influencing them, provide an avenue
for glycosylation engineering to play a role, and for differentiating the glycoprotein
produced from presently available preparations.
9
5. Decision Points in Oligosaccharide Biosynthesis
Glycosylation occurs at particular amino acid residues on a polypeptide - known as
glycosylation sites - as the polypeptide moves from the endoplasmic reticulum through
the Golgi apparatus. However, the oligosaccharide structure attached to a glycosylation
site on a given polypeptide can vary over a large set of chemical and structural diversity
- beginning with the possibility that the site may have an oligosaccharide attached in
some glycoforms but have no oligosaccharide there in other glycoforms (site
occupancy). The polypeptide itself, the host cell, and that cell’s environment all can
influence substantially the distribution of glycoforms produced. The situation is
extremely complicated, making it scientifically and commercially rich in unresolved
questions and opportunities. Unfortunately, there is no way at present to predict what
types of glycoforms will be produced when an arbitrary polypeptide is expressed in a
given host under given conditions (except in the trivial case in which, due to the
polypeptide sequence or the host, no glycosylation occurs). Experience is accumulating
to allow reasonable hypotheses in some cases about how a change from a given
situation will change the glycoform distribution, but, even in this more limited
perspective, many uncertainties remain.
A summary of many of the ways in which glycoforms can differ, with corresponding
examples taken frequently from the set of products listed in Table 1, is provided in Table
3. In some cases, the basic mechanism which determines a particular “decision” in
oligosaccharide biosynthesis can be identified - for example cells will not produce
bisected glycoforms or glycoforms containing sialic acid in to non-
reducing end-galactose residues (found in humans), if they lack, respectively,
or
Different polypeptides expressed in similar hosts under similar conditions can be
glycosylated much differently, clearly indicating that the polypeptide itself exerts a
controlling role for its own glycosylation [3,10,23,24]. A particularly interesting and
technologically relevant example, which also illustrates this connection at the level of
individual amino acid substitutions, is the development of TNK-tPA [25]. A single
mutation, T103N, introduced a new glycosylation site carrying complex-type
oligosaccharides, and at the same time changed glycosylation at native site N l 1 7 from a
high mannose to a complex pattern. This mutant had a 10-fold lower plasma clearance
than wild-type tPA, but only one third of the fibrin binding activity of the wild-type
glycoprotein. This activity could be restored to wild-type level by removing the native
glycosylation site at position N117. The net effect in the resulting double mutant
T103N, N117Q tPA is to shift one site from position 117 to 103 and change the
glycosylation pattern from high-mannose to complex type.
10
6. Tools and Strategies for Manipulating Glycosylation
6.1. CHOICE OF HOST
It is the capability of animal cells to accomplish glycosylation in a manner compatible
with human application which has created a special niche for animal cell technology in
the biotechnology industry. Commonly used bacterial hosts do not glycosylate their
proteins, and yeast glycosylate with high mannose oligosaccharide structures which are
not suitable for injection into humans. Similarly, baculovirus expression systems, as
well as plants, also synthesize oligosaccharides on glycoproteins which are undesirable
for pharmaceutical applications.
Even within the sphere of cultured animal cells and whole animals, different cell lines
and different animals produce different glycoforms following translation of an identical
polypeptide. A few examples of such phenomena are listed in Table 4. A more
extensive list of examples can be found in Reference 11 .
11
6.2. MUTANTS AND METABOLIC ENGINEERING
There are two different classes of genetic manipulation which can influence
oligosaccharide biosynthesis. The first is modification of expression level of enzymes
and related proteins involved in oligosaccharide biosynthesis which are encoded in the
genome of the host cell. There are in turn two alternative technologies for obtaining
host cells with such altered expression levels of its homologous proteins. One, which
has played a critical role in the development of glycobiology, is screening for mutant
hosts which exhibit altered glycosylation. These are typically identified by detection of
the corresponding modification in oligosaccharides displayed on the outer cell surface.
Further information on such glycosylation mutants and their application in
glycobiology can be found in a recent review [2]. It is reasonable to expect that, at least
for some cloned, secreted heterologous glycoproteins, use of glycosylation mutants will
result in an alteration of product protein glycosylation. However, to date there are no
publications confirming this possibility.
A second approach for modifying expression of host cell proteins involved in
oligosaccharide biosynthesis involves several different methods based on recombinant
DNA technology. By gene activation or by installing additional copies of chosen genes
with appropriate transcriptional controllers, particular enzymes can be amplified.
Conversely, antisense and other methods can be employed in order to down-regulate
expression of targeted host cell genes.
In many potential applications of glycosylation engineering, the oligosaccharide
biosynthesis capabilities of the host are extended by introduction into that host of one or
more genes encoding heterologous oligosaccharide biosynthesis enzymes, or possibly
other proteins including precursor transporters, into a heterologous host. This can be
achieved by recombinant DNA methods but not by mutation. A limited number of
experiments employing this approach have so far been published. These involve a
relatively smallset of heterologous enzymes, in part because the working pool of cloned
enzymes for this type of glycosylation engineering, at least in the public domain, is
still small. However, these experiments have so far produced positive and encouraging
results, in the sense that installation of a heterologous enzyme resulted in the
12
corresponding modification in glycosylation for at least a detectable quantity of a
particular glycoprotein which has been analyzed. Table 5 summarizes all such
publications known to the authors as of the submission date of this manuscript.
It is by no means guaranteed that genetic manipulation of the activity of a glycosylation
enzyme will result in a corresponding change or any change at all in the glycosylation
of particular glycoproteins. Failures in this respect are rarely documented, but there is
one published example. Overexpression of cloned in F9
carcinoma cells (which already express this enzyme) gave no detectable change in the
glycosylation of endogenous lysosomal membrane protein 1 (LAMP-1), although the
intracellular level of the enzyme was significantly increased [47].
Glycosylation engineering has also been achieved in a transgenic animal [48J. In this
case, human was trangenically expressed in the
mammary gland of mice. Milk samples from transgenic animals contained soluble,
active forms of the large quantities of a free oligosaccharide product of this
enzyme (2'-fucosyllactose), as well as modified glycoproteins. In contrast, milk from
control animals lacked these glycoconjugates.
Glycosylation engineering to modify glycoforms of a cloned glycoprotein product can be
expected also to influence glycosylation of some set of host cell glycoproteins [49,50],
potentially influencing their function. This could disturb growth, productivity,
development [51], or viability of the host, complicating achievement of the desired
product glycosylation. Engineering global changes in glycosylation capabilities of a
cell, such as modifying a yeast to glycosylate like a human ce11, is extremely difficult
for this and another reason: oligosaccharide synthesis depends not only on the enzymes
directly involved but on synthesis and transport capabilities for nucleoside sugar
cosubstrates and their soluble reaction products, requiring that any missing functions in
this entire complex system must also be installed to enable the new biosynthetic
activity.
As presently understood, oligosaccharide biosynthesis achieves the observed end result
by sophisticated organization and segregation of various enzymes, and the reactions they
13
catalyze, into different organelles. Therefore, achieving synthesis of human
oligosaccharides in a procaryotic cell seems unattainable, whatever new genes and
functions are introduced by metabolic engineering. However, the potential sensitivity of
glycoform distribution to synthesis enzyme localization suggests a new avenue for
manipulating product glycosylation: The enzyme genes can be engineered to alter
targeting of enzymes within the endoplasmic reticulum and Golgi apparatus. Calculated
results indicating the conceptual feasibility of this have recently been presented [52], and
amino acid sequences implicated in targeting proteins among these compartments are
known [53,54], providing essential molecular reagents to undertake such “localization
engineering” (!).
6.3. TECHNOLOGIES FOR EFFECTIVE GLYCOSYLATION ENGINEERING
Success in metabolic engineering depends critically upon availability of a powerful set
of design and implementation tools in order to define potentially effective strategies and
in order to execute them. One area of genetic technology development which is
potentially very important but still relatively undeveloped is antisense methodology in
order to reduce expression of chosen enzymes. Experience with antisense technology and
other applications has shown that it has a greater chance of success if full length
antisense messages are used, although even in this case the extent to which expression
of the targeted protein will be reduced is unpredictable. Antisense technology is only
now beginning to enter the arena of animal cell technology, as presented elsewhere in
this volume.
6.3.1. Antisense Reduction in Cell-Cell-Adhesion for CHO Cells
No prior reports have described successful modification of the oligosaccharides
synthesized by Chinese hamster ovary (CHO) cells by antisense methods. Here such a
study is summarized.
A CHO mammalian cell line that stably expresses FucTVI activity and the cell-surface
was previously engineered. This cell line was transfected with
an antisense-Fuc-TVI full length gene. The FucTVI activity was measured in three
antisense stable cell clones (anti-Fuc-TVI clones) and compared to the and
wild type CHODG44 cell lines (Table 6).
14
The three antisense clones showed different levels of reduced Fuc-TVI activity. The
expression level of in the different clones was therefore investigated (Table 7). An
monoclonal antibody (CSLEX-1) coupled to IgM-FITC was used for the
expression assay.
The expression in the three antisense clones was lower when compared with the
cell line, and null for the wild type CHODG44. As reduced expression
should result in by a lower binding capacity to, for example E-selectin, a simple
adhesion assay of (transfected and untransfected) cell lines to HUVEC cells
(both in the presence and absence of human recombinant which is needed to
induce the expression of E-selectin) was performed. The results are shown in Table 8.
15
Anti-Fuc-TVI clones showed the expected reduced E-selectin binding capacity. These
results indicate that is definitely possible to regulate the expression of the epitope
by antisense technology which specifically targets the fucosyltransferase activity
responsible for its synthesis. This antisense technique can be applied to other
glycosyltranferases to regulate different points of the glycosylation pathway. Antisense
technology of CHO cells, alone or associated with other glycosylation engineering
methods, may bring new possibilities for the production of novel glycoproteins.
6.3.2. Mathematical Models of Complex Glycosylation Pathways
The network of enzyme-catalyzed reactions which conduct oligosaccharide biosynthesis
in animal cells is complicated by two types of general phenomena. First, it is common
for a single species or intermediate in this pathway to serve as a substrate for several
different enzymes, giving raise to correspondingly different further intermediates or
oligosaccharide end-products. Second, certain oligosaccharide biosynthesis enzymes act
on large numbers of different biosynthetic intermediates. These two features render
intuitive or mental simulation of oligosaccharide biosynthesis very difficult. Therefore,
mathematical models which provide a systematic representation of all of the
simultaneous and competitive phenomena involved and which can suggest expected
consequences from the operation of this network, or, more importantly, from
perturbations in it, are indispensable for glycosylation design in the future. A first
model for N-linked oligosaccharide biosynthesis has been presented recently, and its
results show the types of non-obvious qualitative insight which can be obtained by this
approach [52]. The first step of the pathway has also been recently modelled [26]. This
mathematical model incorporates different factors that determine the extent of the
oligosaccharyl-transfer reaction and provides useful insights for the engineering of this
step.
6.3.3. Vectors for Regulated Multicistronic Expression
More sophisticated glycosylation engineering in the future will require coordinated
expression of several sense and/or antisense messages, perhaps at a particular time in a
production process. Recently, convenient multicistronic cloning and expression vectors
for animal cells have been described which can be used in these applications [55].
7. Bioprocess effects on glycosylation
Although it is not the main topic of this review, a few highlights from previous
research exploring interactions between the process environment of a recombinant
animal cell expression system and glycosylation of product proteins may be of interest.
Also, genetic engineering may play a future role in modifying these host cell responses
to environmental changes in a way that makes product glycosylation less sensitive to
these changes or which enhances the consistency and quality of the final recovered,
purified product.
16
Bioprocess effects on glycosylation can be divided into two categories. First, certain
conditions in the bioreactor influence activities within the cell, changing glycosylation
of proteins secreted by those cells. Second, conditions in the bioreactor, and also in
downstream processing, may influence the nature of the end product, including its
glycosylation. It should be noted that the possibility of oligosaccharide modification
after glycoprotein secretion from the cells has not always been considered explicitly in
previous bioreactor studies, so that a rigorous assignment of the phenomena involved as
intracellular or extracellular cannot be made in several cases.
7.1. EFFECTS ON GLYCOPROTEIN BIOSYNTHESIS
Changes in culture conditions such as and ammonia concentration can cause shifts in
the glycoform distribution produced. Several examples which illustrate such phenomena
are given in the Table 9. Mechanisms responsible for coupling between external
conditions and the internal, compartmentalized, enzyme-catalyzed oligosaccharide
biosynthesis reactions which determine glycosylation are not defined, but some
reasonable possibilities have been suggested in some situations. For example, because it
is a weak base and its unionized form is freely diffusible across cellular membranes,
ammonium in the external medium causes changes in intracellular of hybridoma
cells [56,57,58]. This is consistent with, for example, associated changes in sialylation
of O-linked glycosylation of GCSF, via the relationship of the enzyme O-
linked
17
Another bioprocess-glycosylation connection has been elucidated in the elegant study of
Jenkins, James, and coworkers on glycoform distributions of cloned human
produced by recombinant CHO cells [65]. Glycosylation of shifted
significantly as a function time of sampling during a batch cultivation. In terms of
product quality control and process consistency, this work raises profound questions and
challenges for the bioprocess engineer. Such questions could be even more acute for
long-term cultivations with extended times of product harvest, such as fed-batch and
perfusion culture.
7.2. POST-SYNTHESIS EFFECTS
Glycoproteins exit the cell and enter the culture medium where they are exposed to other
proteins secreted by the cells, proteins released from lysed cells, and probably serum
proteins as well (although medium is changed between cell growth and production
phases in several manufacturing processes, this does not completely eliminate serum
proteins, as anyone who has tried to wash away serum proteins by extensive treatment
of the cells in the laboratory before doing SDS-PAGE analysis knows).
Neuraminidase, an enzyme which cleaves sialic acid residues on oligosaccharides, has
been detected at potentially significant levels in recombinant CHO cell cultures, and has
been shown to reduce sialylation of secreted glycoprotein products [66]. Activities of
several oligosaccharide hydrolases
have been detected in insect cell
(Sf9) cultures [67]. These findings invite genetic manipulation to reduce expression of
these product-degrading enzymes [68].
8. In Vitro Oligosaccharide Fractionation, Remodeling and
Biosynthesis
Obviously the processing pathway to the packaged product does not end with the
bioreactor. Different types of technology can be applied downstream to modify
glycoforms in the final product. Simplest among these are separation methods which
fractionate the product into different glycoforms, allowing selective environment of
preferred molecules. Glycoforms can be fractionated to some extent on the basis of
charge or hydrophobicity, while highly specific separations can be achieved using
various lectins (e.g., immobilized on a chromatography matrix) which offer exquisite
selectivities for a wide variety of particular glycosidic linkages [69].
Many enzymes are known, and some commercially available, which synthesize or which
hydrolyze particular glycosidic bonds. These are important tools in current technologies
for oligosaccharide analysis, and they are also potentially useful to accomplish in vitro
modifications of glycoproteins. In the extreme case many or all of the oligosaccharides
on the glycoprotein could be synthesized in vitro. One major barrier to this concept is
18
cost of the enzymes, and especially of the nucleoside sugar donor substrates required for
glycosidic bond synthesis.
For decades organic chemists have dreamed of replacing microorganisms for synthesis of
natural products such as cephalosporin. This remains a dream, but chemistry - including
enzyme catalysis - is a vital contributor in modifying natural products to obtain an
optimized pharmaceutical. It seems likely that a similar scenario will unfold concerning
glycosylation.
9. Concluding Remarks
By modifying oligosaccharide synthesis processes in the production host, glycosylation
engineering interacts with process and polypeptide influences to determine product
glycosylation which in turn can influence many practically important functional
characteristics of that glycoprotein (Figure 1). The future potential of this technology to
contribute to new and improved pharmaceuticals is great. Progress in the field now is
limited by lack of a greater number of cloned genes for enzymes and other proteins
involved in oligosaccharide biosynthesis, by insufficient knowledge about
oligosaccharide structure-glycoprotein function relationships, and by underdeveloped
tools for genetic design and manipulation of glycosylation. However, motivated by the
scientific and commercial importance of this field, rapid progress in all of these areas is
evident, expanding future horizons.
19
10. Acknowledgements
The authors’ research in glycosylation is supported by the Swiss Priority Program in
Biotechnology (SPP BioTech).
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Discussion 23
Lupker:
An important thing about glycosylation is the in vivo function and,
Bailey: in this respect, are you aware of groups trying to make knock-out
mice for some of the glycosylation enzymes?
Lupker: Yes, but there was no possibility to cover this approach in this talk.
Bailey: This will show how host changes can be tolerated and we will
Bernard: probably find large sensitivities.
How many genetic diseases exist in humans that concern
Baily: glycosylation, or are they all lethal?
I do not know the answer to this good question.
You mentioned that we have to be cautious when we produce
targets in baculovirus insect cells. Do you have specific examples
of proteins that we could not study in vitro function because of the
specific glycosylation occurring in insect cells?
No, this is another area where the structure/function data is absent.
There are a few examples of the polypeptide structure depending
upon which glycoform you have. We could question whether de-
glycosylated polypeptides used for X-ray structure analysis, and
subsequent drug design, are the right configurations.
SESSIONS ON :
BIOSYNTHESIS AND POST-TRANSLATIONAL MODIFICATIONS OF
RECOMBINANT PROTEINS, and
CELL PHYSIOLOGY AND METABOLIC ENGINEERING OF ANIMAL
CELLS
Over the last decade the expression of recombinant proteins in animal cells has evolved
from a fine art reserved to specialists to almost a routine business. The focus has changed
from the quantitative aspects of expression to the quality of the protein produced with a
special emphasis on post-translational modifications such as glycosylation. In addition,
considerable interest has now been invested in understanding, modifying and improving
metabolic and synthetic pathways in animal cells.
Despite the relative ease with which many recombinant proteins can be manufactured by
animal cells today, we should not forget that some proteins are extremely difficult to
produce and that, for others production is far from optimal. These problems present the
justification for the continuation of fundamental research on gene expression and
transfection systems.
Of equal importance, as discussed in the papers of the glycosylation chapter, is the effect
of manipulating the environment on post-translational modifications in general, and on
glycosylation in particular. Among other parameters, the carbohydrate sources and
ammonium/glutamine concentrations have been shown to have significant effects.
Considerable interest has now been invested in understanding and modifying (or
redirecting) metabolic pathways in animal cell lines, when this cannot be achieved by
cultural means. The section on metabolic engineering comprises papers dealing with the
possibility for modifying the rapid glycolysis and glutaminolysis that generate high levels
of waste metabolites by overexpressing the glutamine synthetase gene and thus eliminate
the need for glutamine supplementation. Other matters of interest are the modification of
glycosylation (novel glycoforms) of proteins by genetic engineering means. This can be
achieved either by expressing new sugar transferases in cells, or by eliminating certain
transferase activities by an antisense RNA-approach.
Cell proliferation and apoptosis are two interlinked fundamental processes which are
important for the industrial production of drugs. Therefore, these processes must be
fully understood in order to be able to regulate them. On one side, cell growth should be
reduced in order to increase the specific productivity. However, this can lead either to
increased cell death or, in the case of induced growth reduction (IRF-1 fusion protein -
presence of estradiol leads to a growth reduction) to reduced productivity. By using
genetic means, this negative effect can be avoided. The productive cell mass may be
reduced by premature cell death (usually apoptotic) caused by culture stresses.
Different means to reduce or avoid premature apoptosis were pre-sented, based
for instance on the overexpression of bcl-2 or c-jun antisense genes, leading to a
prolonged growth phase and often to an increased productivity. These papers indicate
25
O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 25-26.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
26
that key cell-cycle control, and apoptosis genes and the proteins they encode provide
targets for new approaches to improve large-scale production.
Considering that the main reason for choosing an animal cell production system over
cheaper and easier alternatives is for their ability to process proteins optimally for
bioactivity as therapeutic or diagnostic agents, this area of research is very important. It
should enable culture processes to become more efficient resulting in more economical
and effective bioproducts. Thus the topics of both sessions on Biosynthesis and post-
translational modifications of recombinant proteins, and on Cell physiology and
metabolic engineering of animal cells are very much interrelated, and represent key
areas for the development of efficient and reproducible animal cell technology.
B. Griffiths, J. Lupker, M. Al-Rubeai, and J.E. Bailey
Chairpersons
GENE EXPRESSION
ENHANCED RECOMBINANT PROTEIN EXPRESSION IN INSECT CELLS IN
THE PRESENCE OF DIMETHYLSULFOXIDE (DMSO)
LUCIANO RAMOS, JAMES F. KANE, & AMY A. MURNANE
SmithKline Beecham Pharmaceutical, Philadelphia PA, U.S.A.
1. Abstract
The use of insect cells to express many recombinant homologous and
heterologous proteins is reported throughout the scientific literature. A popular insect
promoter is the metallothionein (Mtn) promoter which can be induced by the addition of
heavy metals such as In this report we present data from Drosophila
melanogaster Schneider 2 (S2) cells that have been stably transfected and express several
different proteins under the control of the Mtn promoter. Selection was achieved using
the hygromycin B-resistant gene derived from Escherichia coli. Our data show that such
stably transfected insect cells expressing several different types of proteins, such as fusion
proteins, soluble receptors and antigens, have enhanced product expression when the cells
are treated with 1% dmethylsulfoxide (DMSO) prior to induction with When
DMSO is added at least 12 hours before the addition of 750 uM protein
expression increased 5 to 8-fold. The addition of DMSO at other times, was not as
effective in stimulating expression. Furthermore, DMSO did not affect expression from
the D. melanogaster constitutive actin promoter.
2. Materials and Methods
Cell lines: Three independent stable cell lines of D. melanogaster cells were selected
following transfection with plasmid DNA encoding a fusion protein (S2-F), a soluble
receptor (S2-R) or an antigen (S2-A) using procedures previously described (4,5). All of
these genes were under the transcriptional control of the D. melanogaster Mtn promoter.
Growth Medium: Growth medium is a proprietary SmithKline Beecham serum-free
insect media designated as SB insect media .
Experimental conditions: Cells were propagated in 250 ml Bellco spinner flasks or
Corning Erlenmeyer disposable 250 ml flasks. Cells were seeded in these flasks by
means of dilution into SB insect media to attain a seeding density of about
in a volume of 100 ml per flask. Cultures were incubated at 24°C on a shaker
base set at 120 RPM. Cell counts were performed using the ZM Coulter counter and
viability assessed using trypan blue dye exclusion. For production studies, cells were
incubated for 3-5 days and induced at a cell density of
and incubated for an additional 3-5 days. For experiments where DMSO was
used, cultures were treated similarly except that DMSO was added to achieve the desired
concentration used in our studies.
29
O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 29-33.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
30
Product assay: Quantitation of product expressed by the various cell lines was
performed using ELISA based assays
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