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

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Bio Project GmbH

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Bio Project GmbH