Escolar Documentos
Profissional Documentos
Cultura Documentos
and
Scale up
for mAbs
Daniel Karrer
Phase 1
Phase 2
Phase 3
Speed to Market
Clinical Batches
Cost is secondary
Robust / Simple
Purity
Validateable
Scale up
Supply Market
Develop Process
approved
Priority
Change
GMP Consistent
Generic Process Model
Economic
Flexibility / Quick
changeover
High throughput / yield
Low running cost / risk
Reduces development
time
Process parameters,
harvest, stability, dosage
Impurity
Culture Fluid Clarification Capture Purification Polishing Virus
Cell Debris
Colloids
DNA
HCP
Virus
Isoforms
Ligand
UF
Culture
Fluid
Production Qty
$/gram
Process/Cycle time
Equipment size
Process Parameters
Cost
Total costs
Fixed costs
Variable costs
Purity/Quality/Yield
Throughput
Cost in k$
10.0
2.0
Buffer Filtration
10.0
4.0
Air
0.8
40 k$/50 batches
4.5
240 m2 Prostak / yr
4
2
4
11
8x 16Millistak / batch
Polysep 9 x 30
Durapore 9 x 30
MF (Prostak)
Depth
Clarification
Sterile
Chromo AF
Ultrafiltration
3.5
8
2
0.3
Chromo IEX
Virus
TOTAL
66.1 k$
Base :
10 m3 batch
100 batches/yr
0.2g/l
Cell
culture
Prim Sep
Clarification
Scale up
Strategy
Manufacturing
Challenges:
Determine which
technologies are economical
Batch
3-7 days
Fed-batch
7-15 days
Perfusion
> 20 days
Solids
Low
<1%
Medium
2-3%
High
>3%
Low
Medium to high
High
<3 x 10 cells/ml
5-15 x 10 cells/ml
>10 x 10 cells/ml
Cell Viability
High
>90%
Medium to High
20-90%
Medium to Low
<50%
Colloids
Low
Medium to High
High
Turbidity
Low
<200 NTU
High
>1000 NTU
Very High
>>1000 NTU
Ease of
Clarification
EASY
MEDIUM
DIFFICULT
Cell Density
Sterilizing Filter
Secondary
(bioburden and colloids)
Depth/Prefiltration
(debris and colloids)
Bioreactor
PRODUCT
Bioburden
Particles
Colloids
Cells
Debris
Fed Batch
Batch
AF
Chrom.
Centrif/Clarif.
(whole cells)
Clarification
(whole cells/cell
debris)
Clarification Technology
High
Disposables
low capital $
Manufacturing
Lab Scale
NFF
TFF
Low
Disposables
High capital $
Centrifuge
< 10 batches/yr,
<1,000 Liters
>> 10 batches/yr,
>>1,000 Liters
SIP/CIP integrated
Low shear forces
Scalable to large scale
NFF clarification:
Filtration Centre:
Flexibility is key
2 Depth
Filter Steps
2 Pre- Filter
Steps
2 Sterile
Filter Steps
20
NTU
25
Increase of SF surface
No automated control
Difficult, costly and time
consuming handling
15
10
5
0
0
Sterile
0.22 micron
as a function vs
of Feed
turbidity
Sterile
0.2
umforCapacity
capacity
Turbidity
Mammalian Cells
10000
l/m^2 = 1322.1(NTU)-1.5022
R2 = 0.8301
1000
L/m^2
Turbidity breakthrough
100
10
0.1
1.0
NTU
10.0
100.0
100
200
L.M-2
300
Secondary Prefiltration:
Millistak HC
High Capacity
Depth Filter
Secondary
Prefiltration
Sterilizing Filter
(bioburden and colloids)
Open grade
Tighter grade
Cellulosic Spin Filter
Membrane (whole cells)
AF
Chrom.
Flow
0.025
Centrifugation
0.020
Standard Millistak
Technology
Multilayer Millistak
Technology
0.015
0.010
(whole cells)
0.005
Clarification
0.000
0
200
400
600
800
1000
0.22 Durapore Throughput (L/m2)
1200
1400
(whole cells)
Increase of throughput
Filtration Centre :
Clarification Step:
Scale up Recommendations
Optimize
Scale with
Purification
Polish
Capture
Benefits
Case:
batch of MAb
dynamic binding capacity 20 g/L resin
10 L bed (25 cm diameter x 21cm bed height)
Increasing capacity :
Prosep-A
70
20 cm, 0.66 cm ID
60
17 cm, 1.1 cm ID
50
5 cm, 0.66 cm ID
5 cm, 1.1 cm ID
40
20 cm,0.66 cm ID
30
20 cm, 1.1 cm ID
10 cm, 1.1 cm ID
20
10
0
0
10
12
14
16
18
20
5000
5000
5 cm, 0.66 cm ID
5 cm, 1.1 cm ID
Productivity (g/hr)
20
Agarose
Agarose (optimised)
15
10
0
0
10
15
20
25
0.30
0.25
0.20
0.20
10 cm
Agarose
CPG
0.10
44 cm
0.10
9 cm
0.05
25 cm
0.00
0
200
400
600
800
Mobile phase velocity (cm/h)
1000
20 cm
30 cm
50 cm
0.15
0.15
0.05
Bed height
P re s s u re d ro p (M P a )
30 cm
0.25
Pressure drop (M Pa)
0.00
1200
500
1000
Mobile phase velocity (cm/h)
1500
Scale-up :
Conventional scale-up
Increase diameter
Constant bed height and linear
flow rate
Linear increase of bedheight and
flow
Pressure-flow limitations due to
compressibility of conv. media
Combined scale-up
Combined
scale-up
Residence time
scale-up
Conventional
scale-up
Scale up:
3000
2500
2000
AU (280nm)
1500
1000
500
0
0
50
100
mg Ab/ml media
Column
diameter
Bed Height
(mm)
Bed volume
(ml)
Residence
time (sec)
Flow velocity
(cm.h-1)
Dynamic capacity
(mg.ml-1)
44
22
16
25
100
190
38
38
38
90
91
91
100
395
750
17.8
17.7
16.2
47 l
Alternative 1
470 l
Alternative 2
470 l
470 l
Scale Up
Method
Media
Volume
(Liters)
Bed
Height
(cm)
Bed
Diameter
(cm)
Production
Rate
(g MAB/hr)
Chromatography
Equipment
Depreciation
Costs ($/batch)
Conventional
470
15
200
726
1500
1.5
Alternative 1
470
30
140
701
800
0.8
Alternative 2
470
45
115
681
600
0.6
PG 700, 1.0 mg/ml MAb, 10 KL batch, 10 yr column life, 300 cycle resin life, 100 batch/yr
200.00
180.00
Cost (k $)
160.00
140.00
120.00
100.00
80.00
60.00
40.00
20.00
0.00
0
200
400
600
800
1000
1200
1400
2'500.00
Cost (k $)
2'000.00
1'500.00
1'000.00
500.00
0.00
0
200
400
600
800
1000
1200
1400
1600
Alternative
Conventional
10 cycles
Bed
Height
(cm)
Bed
Diameter
(cm)
Prod
Rate
(g MAB/hr)
Chromat
Media
Costs
($/column)
Conventional
470
15
200
726
3.76
12500
1.5
Alternative
47
15
63
73
0.37
12500
0.6
Scale Up
Method
Chromat
Media
Costs
($/batch)
Chromat
Equip Cost
($ (Millions))
Capture Step :
Scale up Recommendations
Dynamic Capacity
Combined Scale-up
Chose first media with high dynamic capacity at low residence time
(micro particulate packing)
Optimize for above media bed height at minimal residence time and
acceptable pressure drop
Calculate column diameter needed for full scale
Split batches
Splitting of batch volume to reduce capital cost and risk using one or
more columns of smaller diameter with optimized bed height
Virus removal
Virus Clearance
Virus Filtration
Robust
High Yield
Recovery ca 98%
Fluid
Pressure (psi)
Mab (2.5 g/l)
30
DMEM
30
Mab (2.5 g/l)
45
DMEM
45
PPV LRV
> 4.7
> 6.5
> 5.7
> 6.2
Sizing is
determined by flux
& capacity
High Flux is key for
low process time
at comparable filter
surfaces
Vmax
1
J*t
Vmax
8.0
sqm/100 L
Flux J
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
0
6
hrs process
10
12
8.0
7.0
6.0
5.0
Blue Dextran
4.0
3.0
2.0
1 ppm
100 ppm
1.0
500 ppm
0.0
0 10 20 30 40 50 60 70 80 90 100
Percent Flow Decay
Viresolve NFP
30 psi TMP
Relative Membrane Area Requirements
(m^2 /m^2 optimal)
Vmax ( l/m^2)
900
800
700
600
500
400
300
200
100
0
1
IgG 1
IgG 2
IgG 3
BSA 1
BSA 2
10
100
Concentration (g/l)
Capacity (Vmax) can decline
with protein concentration
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0
10
15
20
UF / DF
50cm
5ml - 1000 ml
Pellicon XL
10kd
Retention
>99.95%
99.9%
Flux
118 lmh
80 lmh
Recirc.
4 lpm
6 lpm
Line Size
1.5
2.5
Hold Up
8.4 l
20.8 l
Yield
Plus 2 3 %
Optimization of Trans
Membrane Pressure
Reduction of process
time
Reduction of
membrane surface
Flux [L mh]
Final (high)
Prot. Concentration
DF Strategy
Concentrate
Diafilter
Permeate
Permeate
Concentrate
Flux
Retentate
Turbidity
Retention
Cg/e
ln(g/L protein)
Cg
UF/DF Step:
final volumes
Thank You
Thank You
Acknowledgments/Reference
Bio
Project GmbH
Project Management
at it's best
Process Design/Optimisation
Scale up
Process Reviews / Economy
Feasability Studies / Technology
assessements
Engineering Concepts
Technology Transfers / Outsourcing