An evaluation of Protein A and Non-Protein A methods for the recovery of monoclonal antibodies and considerations for process scale-up

Martin Smith

LONZA, 224 Bath Road, Slough, SL14DX

Presented at “Scaling-up of Biopharmaceutical Products”, 26/27th January 2004, The Grand, Amsterdam

13 Apr 2004 / 2

LONZA CUSTOM MANUFACTURING

At present, LCM offers production and purification of monoclonal antibodies at two sites

Portsmouth, New Hampshire, USA3*20,000L Reactors, associated purification train2*5000L, 1*2000L, 2*1500L (perfusion)

– associated purification trains

Typically Phase 3 launch & In-market supply

Slough, UKmix of 200 and 2000L reactorsMultiple batch dedicated purification facilities

Typically Phase 1 & 2, rapid speed to clinic/market

13 Apr 2004 / 3

Background

At early phase development, speed to clinic is critical

At all scales of operation, low cost of goods with reliable, scaleable manufacturing operations is essential

Protein A capture widely regarded as fastest route for purification process development

LONZA experience with >100 antibody processes has resulted in generation of truly “generic” approach to antibody purification

13 Apr 2004 / 4

Potential Scale constraints

US, 3*20,000L reactorsFacility designed to handle wide range of current and future monoclonal antibody processes

As fermentation titres increase, need to address impact on downstream requirements

Product and buffer hold tanks

UK, mix of 200, 2000L reactorsFacility designed to be very flexible across scales of operationSeek to maximise facility throughput to increase customer base and speed products to clinic

13 Apr 2004 / 5

Presentation overview

Introduce case study for generic purification process upgrade

Highlight key operational challenges with traditional recovery technology

Demonstrate improvements resulting from process development to a high throughput process upgrade

Examine future potential to improve purification costs.

13 Apr 2004 / 6

Introduction to case study

Case Study, IgG1 from GS-NSO cell line

2,000L reactor at ~0.5g/L, 1kg into purification

“generic” purification process, average capacities, average number column cycles, typical fluxes…etc

PnA – VI - IEX – UF – IEX – VRF – UF – SF

LONZA generic approach traditionally utilises compressible matrices during chromatography operations

13 Apr 2004 / 7

What’s the problem with compressible matrices

0 100 200 300 400 500 600 7000

5

10

15

20

25

30

35

40

Pressure Drop versus Flowrate

Packed bed Height = 15cmMatrix = Sepharose 4 Fast FlowBuffer =PBS @ 22°C

Pres

sure

Dro

p (p

si)

Linear velocity (cm/h)

1.6cm 2.6cm 5.0cm 10cm 20cm 28cm 40cm

ColumnDiameter

d1400

Chromatography scale-up usually achieved by increasing diameter only

Bed height & flow rate kept constantThis reduces supportive wall effectsResults in increased pressure drop at same flow velocity

Imposes severe limits to usable bed heights and flow rates

Stickel JJ, Fotopoulos A.

Biotechnol Prog. 2001 Jul-Aug; 17(4): 744-51.

13 Apr 2004 / 8

Implementing High Productivity Process upgrade

Numerous strategies are available

Simplest route is to upgrade compressible matrices to high flow rate rigid matrices

Should improve schedule but not buffer consumption

Targeted initial two column steps for matrix upgrade evaluation.

Imposed constraint…..chromatography buffer chemistry should remain the same despite change in matrices.

Reflects a typical rapid development project

13 Apr 2004 / 9

Purification schedule constraints

Duration of any purification batch is dependent on many factors.

Binding capacity vs flow rate of chromatography matricesColumn size, aspect ratio

Operational philosophyShift patterns, labour availabilityEquipment availability/turn aroundHold pointsTarget throughput per annum

13 Apr 2004 / 10

Batch productivity

For purposes of case study, duration of typical batch is represented as 100%

Improvements due to upgrades provided as % decrease in overall batch length

Normalised Batch time = 100%

VI UF IEX UF VRF IEX UFPrA

13 Apr 2004 / 11

PnA Sepharose vs Rigid Upgrade

0 10 20 30 40 50 600

20

40

60

80

100 Rigid Upgrade, 0.5g/L

Bre

akth

roug

h (C

/Co)

%

IgG loaded (mg/mL.matrix)

PnA Seph, 0.5g/L

PnA load concentration ~ 0.5g/L representing for example very low fermentation titres

PnA Sepharose DBC=18g/L

Rigid Upgrade DBC=18g/L

However Rigid Upgrade results obtained at 3x higher flow rate

Greater differences exist at higher breakthroughs

But at expense of yield

13 Apr 2004 / 12

Effect of PnA load concentration on PnAcapacity

PnA load concentrations >5g/L result in dramatic increases in PnAdynamic binding capacity

PnA Sepharose DBC=33g/L(83%higher than at 0.5g/L)

Rigid Upgrade DBC=23g/L(23% higher than at 0.5g/L)

Rigid Upgrade results obtained at 3x higher flow rate

Direct impact on throughput… 0 10 20 30 40 50 600

20

40

60

80

100

Rigid Upgrade, 5g/L

IgG loaded (mg/mL.matrix)

PnA Seph, 5g/L

Bre

akth

roug

h (C

/Co)

%

13 Apr 2004 / 13

Effect of PnA upgrade on process throughput

First target for high throughput upgradeImplement rigid matrix for PnA step

Maintained column geometry (& cost)Increased number of cycles (4 to 5)Decreased PnA step time (by 60%)Increase in buffer consumption (by 25%)

Overall productivity improvement of 15%

PrA VI UF IEX UF VRF IEX UF

VI UF IEX UF VRF IEX UFPrA

-15%

13 Apr 2004 / 14

Upgrade IEX productivity

Evaluated impurity and contaminant clearance ofSepharose based IEX step vs.Rigid IEX matrix

Experimentally verified…Equal clearance of contaminants, impuritiesEqual protein purificationIdentical product quality and step yield

Major improvement…3x increase in step productivity due to 3x increase in linear velocity for same column volume.

13 Apr 2004 / 15

Effect on production schedule of PnA and IEX rigid matrix upgrades

PrA VI UFIEX

UF VRF IEX UF

PrA VI UF IEX UF VRF IEX UF

VI UF IEX UF VRF IEX UFPrA 100%

-15%

-23%

23% Productivity increase was achieved using the upgraded high throughput processNo detrimental effect on purification, yield or product quality observed!

13 Apr 2004 / 16

Potential for increased productivity through upgrading 2nd IEX step

Future plans to evaluate high throughput matrices for 2nd IEX step in generic process

Expected to realise similar productivity increases…

Normalised Batch time = 100%

VI UF IEX UF VRF IEX UFPrA

PrA VI UFIEX

UF VRF IEX UF - 44%

13 Apr 2004 / 17

Potential issues arising from use of rigid matrices

Rigid matrices…Generally smaller particles sizes

Slightly higher pressure dropsPacking complicationsShear sensitivityCleaning regimes£/LGeneric applicability to wide range of Mab’s and cell culture feed stocks.

Extensive re-development programs might be required in some cases

13 Apr 2004 / 18

Scale-up Pilot Plant-PurificationSlough UK

HYDRAULIC SKID

PACKING SKID

40cm RESOLUTE COLUMN

55L SLURRY VESSEL

10mm CHROMATOGRAPHY SKID

13 Apr 2004 / 19

LSBO Purification suite P6B

Purification suite P6-B:

1.4m diameter stainless steel columnChromatography skid to left

2m diameter stainless steel chromatography column

Filtration rig to left

13 Apr 2004 / 20

Operational issues implementing high productivity processes

Supply of buffers in a reduced time can shift operational bottlenecks to:

Buffer make-upBuffer holdProduct hold tank sizesPurification suite floor space

(for increase buffer volume per unit time)Subsequent steps

13 Apr 2004 / 21

Disposable buffer makeup and supply capabilities-pilot scale

Buffers prepared 1-2 days ahead of use, in dedicated buffer prep facility using disposable componentsBuffer prep/hold located adjacent to process area.

500L makeup tank 200 and 500L prep tanks with storage vessels

13 Apr 2004 / 22

LSBO Media Preparation tanks

Media Prep

Visible tanks are:1*17,500L

(far left)

2*3,500L(centre/right)

Buffer PrepSimilar in design, vastly increased number of tanks!!

13 Apr 2004 / 23

Reducing buffer make-up by inline dilution

LONZA has successfully implemented in-line dilution for PnA equilibration buffers

Reduced buffer prep requirements by 10x

CIR101 CIR102 AIR121pH FIR141 TIR101 SetMark

0

50

100

150

mS/cm

2.0

3.0

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pH

0.0 2.0 4.0 6.0 8.0 10.0 min

Wat

er ri

nse

1x b

asel

ine

10x_

conc

entr

ate

Feed

back

ena

bled

21.0

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Feed

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13 Apr 2004 / 24

The Story, so far…

Where schedule bottlenecks occur, rigid matrices can dramatically improve individual batch productivities

Scale-up no longer restricted to diameter increases alone

Watch for where the bottlenecks move next…Usually buffer makeup, hold, then product tank sizesAs fermentation titres continue to rise…

Column diameters must inevitably increaseAssociated matrix costs therefore rise

13 Apr 2004 / 25

Non-Protein A recovery processes

Major cost centre for Protein A processes is the cost of the Protein A itself (and CIP reagents)

Currently evaluating numerous non-Protein A alternatives that compete favourably with Protein A wrt

CostDevelopment timelinesPurification (protein, impurities, contaminants)Product qualityOverall process productivity

13 Apr 2004 / 26

Prometic Biosciences-MabSorbent A2P

Provided material to Prometic Biosciences under MTA

Developed two step purification strategy

Fractogel + Mabsorbent A2P

Key benefit is reduced matrix cost per litre(approximately 3-5x reduction in raw material costs)

13 Apr 2004 / 27

Prometic 2 step process for case study IgG

Two step process outline…

1:3 dilution of clarified harvest materialLoad onto S Fractogel (Bind/Elute)Direct load onto MabSorbent A2P

No intermediate UF!

Initial process evaluation stopped after A2P

13 Apr 2004 / 28

Productivity comparison between Protein A and Non-Protein A process

15% Faster

35% Slower

PrA VI UFIEX

S -Fract

VI UF IEXPrA

VI UF VRF IEX UFA2P

Productivity of Prometic two step process is competitive with high throughput PnA upgrade

13 Apr 2004 / 29

Non-Protein A purification performance

Ion exchange antibody capture(Fractogel SO3-)

Mabsorbent A2P polishing step High capacities and flow rates, 150-200 cm/hr)

High recovery (95% 2 step process recovery)High purity (>95%)Contaminant removal (DNA <1pg/mg protein, 2 HCP)No leached affinity ligand detected!

13 Apr 2004 / 30

Conclusions

Capacity constraints in the purification of monoclonal antibody products may be alleviated by employing high throughput rigid matrices.

Increased throughput can be achieved with little increased cost of goods

Capacity bottlenecks shift to other operational areas

Non-Protein A media are becoming competitive with accepted traditional recovery strategies for monoclonal products

13 Apr 2004 / 31

Acknowledgements

LONZASimon Jones

Prometic BiosciencesKeith Watson