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Purification Designand
Scale upfor mAb´s
Daniel Karrer
Scale up Issue: Change in Priorities
Speed to MarketDevelop Process
PurityValidateable
Scale upFast and simple
Clinical BatchesCost is secondary
Development Pipeline
Preclinical Phase 1 Phase 2 Phase 3 approved
Supply MarketRobust / Simple
GMP ConsistentGeneric Process Model
Economic Flexibility / Quick changeoverHigh throughput / yieldLow running cost / risk
Priority Change
"Got a few problems with our linear scale up."
Robust mAb Process:Generic Purification Template
Impurity Culture Fluid Clarification Capture Purification Polishing VirusCell DebrisColloidsDNAHCPVirusIsoformsLigand
mAb´s are sufficiently similar to permit a template approach
Reduces development time
sufficiently different that there always has to be optimization
Process parameters, harvest, stability, dosage
Minimum of steps forhigh overall yield !
UF
CultureFluid
Final Filling
Key for Economic Design/Scale-up in DSP: Low production cost
Throughput = Production Qty
Equipment sizeDrives utilization of equipment and facilities neededDrives fixed cost and headcount
Process ParametersOptimal process design ( min volumes, surfaces and quantities) to reduce cycle timeDrive running/expandable cost
Purity/Quality/YieldDrives batches needed /annum
Process/Cycle time $/gram Total costs
Variable costs
Fixed costs
ThroughputC
ost
Increasing throughput leverages fixed cost
Key for Economic Design/Scale-up in DSP: Know Your Expandable cost
TOTAL 66.1 k$
Expendables/batch Cost in k$ Media filtration (non serum free) 10.0 One time use Capsules (ferm additives) 2.0 One time use Buffer Filtration 10.0 One time use Intermediate bulk sterile filtration 4.0 Air 0.8 40 k$/50 batches
Prim Sep. /Perf. MF (Prostak)
4.5
240 m2 Prostak / yr
Clarification Depth Clarification Sterile
4 2 4
8x 16”Millistak / batch Polysep 9 x 30” Durapore 9 x 30”
Chromo AF 11 100 l over 9 mts (4 x 75 cycles)
Chromo IEX 3.5 2 x 400 l/yr Virus 8 4 x 10” NFP Ultrafiltration 2 75 m2 over 1 yrs Final aseptic filtration 0.3
Base :10 m3 batch
100 batches/yr0.2g/l
Media Prep Buffer Prep
Media and Buffer Preparation Scale-up
Media and Buffer Filtration Scale-up: Performance impacting Economy
Relative Flux of PES membranes Throughput (Filtr. Capacity) of Filters
High flow / high capacity filters neededSuperiority of PES membranes for large volume filtrations
Double flux due to composite asymetric membraneLarger capacity due to second layer for plugging streamsSterility assurance, low prot binding and caustic stability
Multiple use (buffer filtration)Balance expandable cost with utility cost (WFI, Steam)
Cell culture Prim Sep
Clarification
Media prep
Cell Culture and Clarification Scale-up
Clarification Scale-up / Design Strategy
SeparationTechnology : Determines at which scale process can work
Manufacturing Challenges:Determine which
technologies are economical
Scale up Strategy
Cell Culture Type:Determines which clarific.
technologies will work
Mammalian Cell Culture Types
Affecting downstream separation design
CultureType
Batch3-7 days
Fed-batch7-15 days
Perfusion> 20 days
Solids Low <1%
Medium 2-3%
High >3%
Cell DensityLow
<3 x 106 cells/mlMedium to high
5-15 x 106 cells/mlHigh
>10 x 106 cells/ml
Cell Viability High>90%
Medium to High20-90%
Medium to Low<50%
Colloids Low Medium to High High
Turbidity Low<200 NTU
High>1000 NTU
Very High>>1000 NTU
Ease ofClarification EASY MEDIUM DIFFICULT
Clarification Train Technologies:Impact of Cell Culture Type
Bioreactor PRODUCTBioburdenParticlesColloids
CellsDebris
Sterilizing Filter(bioburden and colloids)
Secondary Depth/Prefiltration(debris and colloids)
PrimarySeparation
AFChrom.
Perfusion
Batch
Fed Batch
Spin Filter (whole cells)
Centrif/Clarif. (whole cells)
Clarification (whole cells/cell
debris)
Clarification TechnologyManufacturing Challenge: Economy
Batch Size and Frequency
< 10 batches/yr, <1,000 Liters
>> 10 batches/yr, >>1,000 Liters
NFF
TFF
Centrifuge
High Disposables low capital $
Low Disposables High capital $
Primary Clarification Step
Lab Scale Manufacturing
Perfusion Technology: Large scale
MF 60 m2 media perfusion system- SIP/CIP integrated- Low shear forces- Scalable to large scale
TFF Clarification : Prostak MF
Micro filtration unit: 280 m2, for 12m3 Batch• excellent filtrate quality• up to 99.5 % cell removal• up to 800 g/l wet weight• possibility of cell washing• easy scaling up
• reduced final filtration needs
Secondary Clarification Methods
NFF clarification:Adsorptive Depth Filters ( Pad Filters) with high dirt load capacityPleated Prefilters
Large amounts of colloids limit pleated filter capacity
Staged approach requiredTypically two or three stages
Filtration Centre:Flexibility is key
2 DepthFilter Steps
2 Sterile Filter Steps
2 Pre- Filter Steps
For multitechnology / multiproduct facility
Filtration Centre :Design Concept
Adsorptive depth filters
Current issuesPremature particulate breakthrough after 1st step
Increase of SF surface
No automated controlDifficult, costly and time consuming handling 0
5
10
15
20
25
0 100 200 300
L.M-2
NTU
Turbidity breakthrough
Sterile 0.22 micron Capacity as a function of Feed turbidity for Mammalian Cells
l/m^2 = 1322.1(NTU)-1.5022R2 = 0.8301
10
100
1000
10000
0.1 1.0 10.0 100.0NTU
L/m
^2
Sterile 0.2 um capacity vs Turbidity
Secondary Prefiltration:Filter Train Optimization: Elimination of filtr. step
Sterilizing Filter(bioburden and colloids)
Secondary Prefiltration
(debris and colloids)
PrimarySeparation
AFChrom.
Guarded Sterilizing Filter(bioburden and colloids)
Clarification (whole cells)
Centrifugation (whole cells)
Spin Filter (whole cells)
Open grade
Tighter grade
Cellulosic Membrane
Millistak HCHigh Capacity Depth Filter
Flow
0.22 Durapore Throughput (L/m2)
0.000
0.005
0.010
0.015
0.020
0.025
0 200 400 600 800 1000 1200 1400
0.22
Dur
apor
e R
esis
tanc
e (P
SID
/LM
H)
Standard MillistakTechnologyMultilayer Millistak Technology
0.22 Durapore Throughput (L/m2)
0.000
0.005
0.010
0.015
0.020
0.025
0 200 400 600 800 1000 1200 1400
0.22
Dur
apor
e R
esis
tanc
e (P
SID
/LM
H)
Standard MillistakTechnologyMultilayer Millistak Technology
0.000
0.005
0.010
0.015
0.020
0.025
0 200 400 600 800 1000 1200 1400
0.22
Dur
apor
e R
esis
tanc
e (P
SID
/LM
H)
Standard MillistakTechnologyMultilayer Millistak Technology
Sterile Membrane Capacity Optimization of filtration parameters
Combined flow/pressure controlIncrease of throughput
Karl Schick/SciLog Middleton/WI
Filtration Centre :Flexible Design Concept
Autom. Filter Train
2-3 stage filtration
Flexible design (serial/parallel op)
CIP/SIP automatedIntegrated Integrity TestingFully automatedAlgorithms for pressure/flow/turbiditycontrolFast change overMultipurpose / Multiproduct design
Clarification Step: Scale up Recommendations
Know important feedstock variablesTurbidity, Packed cell volume, Viability, Filterability (Vmax/Pmax/Tmax)
OptimizeAs multistep integrated process
Scale withConsistent culture age and hold time windowsConsistent centrate residence timesConsistent feed volume/filter areaFeed representative for large scale or worst case operation
Good clarification guarantees long lifetime of capture media and keeps media cost under control
Capture
Media prep
Down stream processing Scale-up
Purification Polish
Capture Step: Affinity Protein A
BenefitsHigh purity in one generic step (>98%)High capacity - low media and buffer usageHigh throughputRapid equilibration min. sample lossesLifetime: 300 cyclesPotential for viralreduction/inactivation
Capture process limitationsCapture process limitations
Case:batch of MAbdynamic binding capacity 20 g/L resin10 L bed (25 cm diameter x 21cm bed height)
1993: 0.1 g/L Expression in 2000 LFlow rate 600 cm/hr (PROSEP A): load time 6.8 hours
Process step is volume limited: need high flow
2003: 1.0 g/L Expression in 10 x 200 LFlow rate 600cm/ hr (PROSEP A): Load time 40 minutesProcess step is capacity limited: need high capacity
Increasing capacity :Increase resin surface area of CPG
5000 Å 5000 Å
1000 Å Pore Diameter PGProsep-A
700 Å Pore Diameter PGProsep Ultra®
Protein A porous glass (PG) supportNarrow pore size distributionProtein A immobilized onto PG support
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12 14 16 18 20
Residence Time (min)
Dyn
amic
Cap
acity
(mg/
cm3 )
20 cm, 0.66 cm ID17 cm, 1.1 cm ID5 cm, 0.66 cm ID5 cm, 1.1 cm ID20 cm,0.66 cm ID20 cm, 1.1 cm ID10 cm, 1.1 cm ID5 cm, 0.66 cm ID5 cm, 1.1 cm ID
Dynamic Capacity (10% BT) (1.0 mg/cm3 hIgG feed
McCue, J.T., et.al., J Chromatogr A, 989 (2003) 139
Capacity and Throughput
Productivity vs Residence Time
0
5
10
15
20
25
0 5 10 15 20 25
Residence time (min)
Pro
duct
ivity
(g/h
r)
PAHC
PAHC (optimised)
Agarose
Agarose (optimised)
Consider throughput as well as capacity !!at least 2 x capacity needed to compensate double residence time
Increasing Throughput (Flow):Pressure-flow characteristics
Agarose has relatively higher back pressureIncreasing slope shows compressibility (wall effects)Back pressure varies with column diameter
CPG has relatively low back pressureConstant slope shows incompressibilityBack pressure insensitive to column diameter
0.00
0.05
0.10
0.15
0.20
0.25
0 500 1000 1500Mobile phase velocity (cm/h)
Pres
sure
dro
p (M
Pa)
20 cm30 cm50 cm
Different bed diameters 44 cm bed diameter CPG
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0 200 400 600 800 1000 1200Mobile phase velocity (cm/h)
Pres
sure
dro
p (M
Pa)
Agarose CPG
30 cm
25 cm
44 cm 9 cm
10 cm
Bed height
CPG has a relatively low back pressure even at 50 cm bed and 800 cm/h
Pressure-flow constraint not as limitingAllows for greater flexibility in column selection
Residence timescale-up
Conventionalscale-up
Combinedscale-up
Scale-up :Methods for more flexibility
Conventional scale-upIncrease diameterConstant bed height and linear flow rate
Residence Time scale-upLinear increase of bedheight and flowPressure-flow limitations due to compressibility of conv. media
Combined scale-upModern media are incompressible and maintain a low pressure dropPermits scale up in any dimension at constant residence time
Greater flexibility for equipment
Scale up:Constant residence time
Columns with same bed volume and different bed dimensions
Same dynamic capacitiesOverlapping chromatograms 0
500
1000
1500
2000
2500
3000
0 50 100mg Ab/ml media
AU (2
80nm
)
VL16, 19cm bed
VL22, 10cm bed
VL44, 2.5cm bed
Column diameter
Bed Height (mm)
Bed volume (ml)
Residence time (sec)
Flow velocity (cm.h-1)
Dynamic capacity (mg.ml-1)
44 25 38 90 100 17.8 22 100 38 91 395 17.7 16 190 38 91 750 16.2
Scale Up Pilot Column by 10x to a Bed Volume of 470 litres
Conventional
Example of a combined column scale-upAlternative 1 Alternative 2
0.660068111545470Alternative 2
0.880070114030470Alternative 1
1.5150072620015470Conventional
Initial Chromatography Equipment Cost
($ (Millions))
Chromatography Equipment
Depreciation Costs ($/batch)
Production Rate
(g MAB/hr)
Bed Diameter
(cm)
Bed Height (cm)
Media Volume (Liters)
Scale Up Method
PG 700, 1.0 mg/ml MAb, 10 KL batch, 10 yr column life, 300 cycle resin life, 100 batch/yr
47 l 470 l470 l470 l
Capital Cost columns
Exponential cost increase with diameter
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
200.00
0 200 400 600 800 1000 1200 1400
Column Diameter (mm)
Cos
t(k
$)
Isopak column stainless steel
Media Cost in columns
Exponential cost increase with diameter
0.00
500.00
1'000.00
1'500.00
2'000.00
2'500.00
0 200 400 600 800 1000 1200 1400 1600
Column Diameter (mm)
Cos
t(k
$)
Prosep A affinity media
Scale Up Pilot Column by 10x to a Bed Volume of 470 litres
Conventional
Example of a Column Scale Up: Split batch
12500
12500
ChromatMedia Costs
($/batch)
0.60.3773631547Alternative
1.53.7672620015470Conventional
ChromatEquip Cost($ (Millions))
ChromatMedia Costs
($/column)
ProdRate
(g MAB/hr)
Bed Diameter
(cm)
Bed Height (cm)
Media Volume (Liters)
Scale Up Method
PG 700, 1.0 mg/ml MAb, 10 KL batch, 10 yr column life, 300 cycle resin life, 100 batch/yr
10 cycles
Alternative
Tradeof of time against cost and risk
Optimised Hardware Designs
Compact modern valve designsreplace 3 -way valves with modular valve clusters
Combined bubble trap/filterdecreases dead volumes, piping and improves residence time and yields
Capture Step :Scale up Recommendations
Dynamic CapacityChose first media with high dynamic capacity at low residence time (micro particulate packing)
Combined Scale-upOptimize for above media bed height at minimal residence time and acceptable pressure dropCalculate column diameter needed for full scale
Split batchesSplitting of batch volume to reduce capital cost and risk using one or more columns of smaller diameter with optimized bed height
Media prep
Down stream processing Scale-up
Virus removal
Virus Clearance
Virus FiltrationTrend to Sgmall Virus Filltration
- Parvo Virus removalReliable- Removal based on size
– High LRVs (≥ 4-6) readily achievable
• Robust– Viral clearance insensitive to
operating conditions (pressure,Temp)
• High Yield– Recovery ca 98%
• Short Cycle time
Fluid Pressure (psi) PPV LRVMab (2.5 g/l) 30 > 4.7DMEM 30 > 6.5Mab (2.5 g/l) 45 > 5.7DMEM 45 > 6.2
Viresolve© NFP( 72 L/M2, disks)
Virus Filter Sizing on Flow or Capacity ?
Sizing is determined by flux & capacity
High Flux is key for low process time
at comparable filter surfaces
0.01.02.03.04.05.06.07.08.0
0 2 4 6 8 10 12
hrs process
sqm
/100
L
Filter A: 1000 L/sqm, 15 LMH
Filter B: 200 L/sqm, 100 LMH
Vmax Flux J
m2 = 1 + 1
L Vmax J*t
Virus Filter :Sizing on Capacity
LRV correlated with flow decay (Hirasahi, Kempf)
Same behavior with other plugging agents
Not expected for adsorption
High LRV maintained, using clean feedstock's (Prefiltration)
0.01.02.03.04.05.06.07.08.0
0 10 20 30 40 50 60 70 80 90 100Percent Flow Decay
PhiX
174
LR
V
1 ppm100 ppm500 ppm
Blue Dextran
Dont exceed 50% flow decay(75% Vmax) when scaling-up
Safety margin needed
Virus Filter : Influence of Protein Concentration
Capacity (Vmax) can decline with protein concentration
Vm
ax (
l/m^2
)
Concentration (g/l)
30 psi TMP
0100200300400500600700800900
1 10 100
IgG 1IgG 2IgG 3BSA 1BSA 2
Viresolve NFP Relative Membrane Area Requirements as a function of Protein Concentration
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
0 5 10 15 20IgG Concentration ( g/l)
Rel
ativ
e M
embr
ane
Are
a R
equi
rem
ents
(m
^2 /m
^2 o
ptim
al)
Area & Cost are minimal at concentrations of 8-10 g/l
UF / DF
Media prep
Down stream processing Scale-up
50cm²5ml - 1000 mlPellicon XL
0.1, 0.5, 2.5m²200ml - 100’s litersPellicon Cassettes
Process Scale Holder1 -80 m²5 - 1000’s liters
Pellicon UF Cassettes Scale-up:An example of true linear scalability
Automated UF/DF 15m2 System
Biomax 10Conv. Polysulfon10kd
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 %
Pellicon High Performance Membranes:Reduction of Process Time and Holdup Volume
UF/DF operating parameters:Optimal TMP for process time reduction
Optimization of Trans Membrane Pressure
Reduction of processtimeReduction of membrane surface
Transmembrane Pressure [bar]
Flux
[L m
h]
Initial (low )Prot. Concentration
Final (high) Prot. Concentration
Optimum OperatingPoint
Opt. TMP for Retained ProductsRun at ‘knee’ of flux curve at highest concentrationReduced aggregation, fouling, cleaning issues
ln(g/L protein)
Flux Turbidity
CgCg/e
Retention
Concentrate
Buffer
ConcentrateDiafilterFeed Retentate
PermeatePermeate
DF StrategyReduction of process time and membrane surface
Optimal Diafiltration PointAt Cdiaf = Cg/e ~ 80 - 100 g/L, Minimal Filter Area/Process TimeAt Higher Conc. : Lower Buffer Use, but low
FluxAt Lower Conc. : High Buffer use, high Flux,
less Aggregate Formation
UF/DF Step:Scaling Difficulties HW / Design related
UF/DF Step Challenges :
Trend to very high protein endconcentrations(200 g/l) for liquid formulations, requesting lowest possible holdup volumes
Reachable Endconcentration / Purity : depends on overall design/membrane surface
DF volumes needed can be impacted bysystem design (mixing)
Higher viscosities creating difficulties (150 CP) Elevated temperatures to be used to lower viscosity.
Aggregation/foaming issues impotant for tank/pump design
Product recovery is key (1% yield = 0.5 Mio$ for 10 kg -batch)
UF/DF System : integrated designs for low final volumes
Integrated tank/pump/holder design reducing hold-up volume
Instrument blocks to reduce pipinglength
Modular holders for Flexibility
Process Development Summary
Key elements for Scale-up:
Visualize full scale process and scale down first
Visualize all process steps : integrated approach
Dont forget productivity,yield and economy already at development scale
10 m3 Batch value 40 Mio $ (10 kg protein)
Make your experiments at small scale and the profits on large scale!!
Thank You
Acknowledgments/ReferenceGlen Kemp, MilliporeHerb Lutz, MilliporeKarl Schick, SciLogFred Mann, MilliporeDuncan Low, Amgen
Thank You
Process Design/OptimisationScale upProcess Reviews / EconomyFeasability Studies / Technology assessementsEngineering ConceptsTechnology Transfers / Outsourcing
Project Management at it's best
Bio Project GmbH
Project Management at it's best
Bio Project GmbH
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