Biotech Cell Culture Development
John Thrift
Overview
• How do you make a cell line that expresses a
therapeutic protein?
• Production process for biotech proteins
• Cryopreservation of cell lines
• Cell culture media
Biotechnology
“Old” School Biotech
“New” School Biotech
Millions of years of evolution
Humans, over the millennia, select for
“brew” traits
Several months to genetically engineer
Selected for expression of protein of interest
Yeast cells for beer
Hamster cells for therapeutic proteins
Both are fermentation products – both may be needed for good health !!
Making of an Expression Cell Line
How do you make a cell
line that produces a
therapeutic protein?
Genetic recombination
Using a series of “cut
and paste” operations,
new genetic material
(say human growth
factor) can be inserted
into a DNA construct
(vector) and then this is
used to deliver the gene
into mammalian cells.
rFVIII
Inserting DNA into a plasmid vector
Expression Cell Line Production Overview
Vector =
Gene for protein
of interest +
resistance
Low concentration
amplification
+
selection
Transfection –
internalizes vector
~20 million cells at a time
sub-cloning
Making of an Expression Cell Line - devices
96-well plates for single
cell cloning
ClonePix
Flow Cytometer
Expression Cell Line Production Overview
Now that we have our cells, and the DNA for the protein
is integrated into their genome, what do the cells do ⇒
they make product
n
Where does all this biology take place at an industrial scale ⇒ in well
controlled fermentation systems
Fermentation Process
Currently two fermentation methods predominate
• Fed-batch – Stirred tank with feed
• Perfusion – Continuous process with medium
addition and harvest withdrawal
Fed-batch –stirred tank with feed
100
18
90
16
80
Feed
14
Percent Viability
Million Viable Cells
Fed-Batch Viable Cell Density and Viability
20
70
12
60
10
50
8
40
6
30
4
20
2
10
0
0
0
2
4
6
8
10
12
14
16
Day
Fed-Batch Product Titer
5000
4500
Product Titer
4000
3500
3000
Harvest
2500
2000
1500
1000
500
0
0
2
Inoculate
4
6
8
Day
10
12
14
16
Perfusion – continuous process with medium addition and harvest withdrawl
Cell retention
device
100
45
90
40
80
35
70
30
60
25
50
20
40
15
30
10
20
5
10
0
0
0
20
40
60
80
100
120
Day
Peerfusion Product Titer
1000
900
Short residence time
Product Titer
800
700
600
500
400
300
200
100
0
0
20
40
60
Day
80
100
120
Percent Viability
Million Viable Cells
Perfusion Viable Cell Density and Viability
50
Scale-up process flow - Start small, expand to big
10-L
100-L
1,000-L
10,000-L
Scale-Up
Biotech Process
Fermentation
Harvest
UF
Cell Sep
UFTCF
Freeze
Isolation
Media
Large scale
Storage
Purification
Thaw
C1
VI
C2
C4
C3
C5
Freeze
drying
Bayer
Final
Container
UF
DF
Formulation
Bayer
Bayer
Bayer
Bayer
Bayer
Bayer
Bayer
Bayer
Bayer
Filling
Cryopreservation of Cell Stock is Critical
How are cell stored for future batchs ?
• Cryopreservation - Cells frozen in liquid nitrogen
(-196°C) can be stored indefinitely. Thawing
frozen cells is the first step in the production
process.
Cell banking assures:
The cell line is preserved
The integrity is maintained
Sufficient and consistent source of production cells
is reliably available for the lifetime of the product
Cryopreservation of Cell Stock is Critical
Cryo-Vials (1 - 10 mL)
Typical Cryo Storage Tanks
Cryo-Bags (50 - 100 mL)
Boxes to store 1 - 2 mL vials
Cell culture media
Food (media) in
Protein product out
Feed Me !!
Cell culture media
Cell Culture Medium….What’s in it ? Mammalian cells
require a more complex medium than yeast
• Water - Super high quality - “WFI”
• Glucose - Energy substrate
• Amino Acids - Building blocks of protein
• Salts and trace elements - Osmo, cofactors, mem. pot.
• Vitamins - Required for many biochemical reactions
• Lipids - precursors, cell membrane
• Buffers - maintain pH
• Protein, including hormones and growth factors
Cell culture media
Cell culture medium
DMEM/F12
COMPONENT
SALTS
KCl
MgSO4 (anhyd)
NaCl
NaH2PO4 H2O
MgCl2 6H20
Na2HPO4 (anhyd)
CuSO4 5H2O
FeSO4 7H2O
ZnSO4 7H2O
Fe(NO3)3 9H2O
SeO2
CaCl2 (anhyd)
g/L
0.31182
0.04884
6.1234
0.0625
0.061
0.07099
0.00000125
0.000417
0.000432
0.00005
0.00000222
0.11665
6.8
(L) AMINO ACIDS
L-Alanine
L-Arginine HCl
L-Asparagine H2O
L-Aspartic Acid
L-Cysteine HCl H2O
L-Cystine 2 HCl
L-Glutamic Acid
L-Glutamine
L-Histidine HCl H2O
L-Isoleucine
L-Leucine
L-Lysine HCl
L-Methionine
L-Phenylalanine
L-Proline
L-Serine
L-Threonine
L-Tryptophan
L-Tyrosine 2Na 2H2O
L-Valine
L-Glycine
Cell culture medium
0.01336
0.27435
0.0225
0.01995
0.01756
0.06256
0.02206
0.73
0.07348
0.1057
0.11096
0.16385
0.03224
0.06748
0.02875
0.03676
0.10156
0.01902
0.10771
0.09866
0.02626
2.1
VITAMINS
Biotin
Choline Chloride
D-Ca-Pantothenate
Folic Acid
Hypoxanthine 2Na
Inositol
Niacinamide
Pyridoxine HCl
Riboflavin
Thiamine HCl
Thymidine
Vitamin B-12
Pyridoxal HCl
Putrescine 2HCl
OTHER
Glucose
Sodium Pyruvate
Phenol Red Na
L-Glutathione
Linoleic Acid
Lipoic Acid (Thioctic acid)
Ethanolamine HCl
2-Mercaptoehtanol
Phosphoryl-Ethanolamine
0.001
0.00998
0.00312
0.00366
0.0027
0.01451
0.00302
0.000031
0.000319
0.00317
0.000364
0.00068
0.003
0.000081
0.05
1
0.055
0.0069
0.001
0.000042
0.000103
0.0012
0.00039065
0.0014
4.1
Bayer Berkeley: rFVIII made in 1 year
Pyramid
Berkeley: beer
made
in 1inmonth
??
Bayer Berkeley:
rFVIII
made
1 year
Thanks for your attention
Questions?
Fermentation Process