strategies for the purification of high titre, high volume

Strategies for the purification of high titre, high volume mammalian cell culture batches

Martin P. Smith.

LONZA Biologics plc, 228 Bath Road, Slough, SL1 4DX.

Presented at,Recovery & Purification.BioProcess International European Conference and Exhibition.Berlin, April 2005.

15/04/2005 / 2

Overview

Review of Fermentation process productivity increasesCurrent status

Challenges presented by high titre, high volume batches

Strategies available for dealing with high-titre, high-volume batches

Opportunities within current downstream processing technologiesChromatographyBuffer supplyUltrafiltration

Importance of timely process developmentSimple tools & methods for process development and scale-up

15/04/2005 / 3

LONZA Biologics manufacturing capacity

LONZA currently operates monoclonal antibody facilities in both the US and UK

UK Manufacturing-focused on rapid supply of clinical phase materialVarious disposable units (Wave)2x2000L ALF2x200L ALFAdditional capacity planning exercise underway

US Manufacturing-focused on large-scale late phase & in-market supply

2x5000L ALF1x2000L ALF2x1500L perfusion (dedicated)3x20,000L STR growing to 4 in 2006

15/04/2005 / 4

Biopharmaceutical Production Challenges

Increased demand for cGMP manufacturing capacity at all scales.

Increase in product approvals and marketing extensions driving ademand for large scale capacity (>5000L reactor volume)

Strong demand for small scale capacity driven by full customer pipelines and need for early phase toxicology & clinical study material.

Growing political and ethical pressures to control and reduce drug development time-to-market and production costs.

Cell Line Construction cGMP

Cell Banking

Process Development

Scale-Up &Pilot Prod.

cGMPManufacture

15/04/2005 / 5

Advances in Mammalian Cell Culture Process Productivity

0 5 10 15 20 25

1000

2000

3000

4000

5000

6000

Iteration 1 (22H11) Iteration 2 (22H11) Iteration 2 (LB01) Iteration 3 (LB01) Iteration 4 (LB01) Iteration 5 (LB01) Iteration 5 (CY01)

Pro

duct

Acc

umul

atio

n (m

g/L)

Fermentation Time (days)

Fermentation productivity is rising RAPIDLY.

Advances in cell culture realising higher titres

Already experiencing 4-5g/L batches after 14days in cGMP reactors

15/04/2005 / 6

Current Capacity Crunch

Much of today’s production capacity for in-market supply designed for low to modest productivities of monoclonal antibodies

Resulted in requirement for multiple large volume reactors (4-6 x 12-20kL) for single purification trains.

In-house BioPharma capacity can be optimised for specific medium to low risk products

Contract Manufacturing capacity must remain reflective of wider industry status to capture current and near term business

Rate of technology development in mammalian expression outpacingdesign-and-build timelines for new facilities

Industry requires low risk, low capital and immediate solutions to purifying high productivity batches across all scales.

15/04/2005 / 7

Identifying bottlenecks in downstream processing

Situation for CMO’s is more complicated than single product facilities

Business risk managed through large customer base

Each product differs widely despite generic/platform technologies

Fermentation productivitiescolumn capacities, number of column stepsDiffering degrees of process intensification

Increasing titres poses significant challenges for:

Throughput (speed of purification)Economics (Batch cost, cost of goods)

15/04/2005 / 8

Strategies to alleviate downstream processing bottlenecks

1. Invest in development of novel technologies

Membrane Adsorbers (PnA capture and contaminant removal)Mimetic ligandsMonolithic chromatography supportsUV for virus inactivation

2. Invest in development of conventional technologies

Centrifugation2 phase separationsSolvent extractionPrecipitationCrystallisation

15/04/2005 / 9

Factors complicating selection of strategy

5g/L titres are here now!

“Novel” technology and re-development of “conventional” technologies may not help in the near term

Both involve significant investment of capital to refit existing facilities

Need to “sell” technology improvementsInternally across companyExternally to clients

Technology solutions must be scalable and generic

15/04/2005 / 10

3. Maximise utilisation of existing assets.Process OptimisationDownstream Yield Improvement ProgramsProcess IntensificationReduce processing timeEliminate non-processing timeReduce start-up and turn around/change over times

4. Drive process excellence across businessLEAN/Six Sigma/Quality systems

Further Strategies to alleviate downstream processing bottlenecks

15/04/2005 / 11

0.1-1g

Constructexpression

vector

ConstructCell Line

DevelopManufacturing

Process

Non GMPPilot Run

GMPProduction

Cell culturesupernatants

Protein Apurifiedproduct

Researchgrade fully

purifiedproduct

Material fortoxicologystudies,

referencestandard,

stability studies

Clinical TrialSupply

MaterialSupply

TypicalQuantities 10-50mls 10-50mgs 10-100g

0.1kg tomulti-kgs

Typical Development Program

Speed to Clinic for Early Phase material ensured through use of generic processesFast Track Downstream development characterised by aggressive timelines

15/04/2005 / 12

Focus of presentation

Bottlenecks and Production Economics change markedly as a function of scale.

Key bottlenecks and areas for cost/throughput optimisation:

Protein A Capture

Buffer Supply

Ultrafiltration Optimisation

15/04/2005 / 13

Example of 2000L reactor titre increase.

For same purification process:

Chromatography elution tank volume constraints

Ultrafiltration tank volume capacity

Virus filtration throughput constraints

Increased demand for buffers

Effect of fermentation titre on intermediate process volumes

Process Volume vs Concentration

0

500

1000

1500

2000

2500

Har

vest

CC

S

PnA

Elu

ate

VI E

luat

e

UF

1Pr

oduc

t

Q F

T

UF

2Pr

oduc

t

VR

FPr

oduc

t

SP P

rodu

ct

UF

3Pr

oduc

t

Pro

cess

Vol

(L)

0

5

10

15

20

25

30

Proc

ess

conc

(g/L

)

Lg/L

Process Volume vs Concentration

0

500

1000

1500

2000

2500

Har

vest

CCS

PnA

Elu

ate

VI E

luat

e

UF

1P

rodu

ct

Q F

T

UF

2P

rodu

ct

VR

FP

rodu

ct

SP

Prod

uct

UF

3P

rodu

ct

Pro

cess

Vol

(L)

0

5

10

15

20

25

30

Proc

ess

conc

(g/L

)

Lg/L

1g/L

5g/L

15/04/2005 / 14

Process Development Case Study 1

Effect of Fermentation Titre on Buffer Demand

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

1 2 3 4 5Fermentation Titre (g/L)

Tota

l Buf

fer D

eman

d (L

)

Reducing Buffer loading during downstream processing

10-15 % of buffer Demand is derived from Protein A step

2000L reactor titre increase.

15/04/2005 / 15

Large Scale Build Out Facility,Buffer Hold-Upper Level

15/04/2005 / 16

Buffer Concentrates and In-Line Dilution

Possible to minimise Buffer Make-up and Hold requirements through in-line dilution

Example: Protein A Equilibration, Post Load Wash Buffer– 50mM Glycine Glycinate, 250mM Sodium Chloride, pH 8.0

C

H

HH2N

COO-Na+

C

H

HH3N+

COO-

C

H

HCl-H3N+

COOH

Glycine Hydrochloride

(AA+1)

Isoelectric Glycine

(free base)

(AA0)

Sodium Glycinate

(AA-1)

pKa1=2.35 pKa

2=9.78

Implementing in-line dilution requiresIn-depth understand of buffer chemistrySolubility and stability of buffer concentratesEffect of temperature on pH and mS/cmAppropriate equipment for in-line dilution

15/04/2005 / 17

Protein A Equilibration Buffer Chemistry

5 10 15 20 25 307.85

7.90

7.95

8.00

8.05

8.10

8.15

8.20

8.25

8.30

pH (-

)

Temperature (°C)

16

18

20

22

24

26

Con

duct

ivity

(mS

/cm

)

0 2 4 6 8 107.85

7.90

7.95

8.00

8.05

8.10

8.15

8.20

8.25

8.30

pH (-

) Concentrate Strength (-)

0

20

40

60

80

100

120

140

160

Con

duct

ivity

(mS

/cm

)

Effect of temperature on at-strength buffer pH & mS/cm

Static dilution of buffer concentrates

Establish process tolerance for pH and mS/cm (as wide as possible)

Check ability to control concentrate and WFI temperature

Ensure buffer concentrate is soluble at required strength

Check Salt Strength at pH for compatibility with MOC’s

15/04/2005 / 18

0 20 40 60 80 100 1200

102030405060708090

100

% B

Volumetric Flowrate (L/h)

10x Dilution

5x Dilution

Demonstrating potential for in-line dilution

15/04/2005 / 19

0 20 40 60 80 100 1200

102030405060708090

100

% B


10x Dilution

5x Dilution

10x Concentrate dilution at 20L/h

CIR101 CIR102 AIR121pH FIR141 TIR101 SetMark

0

50

100

150

mS/cm

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

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

10x concentrate: 500mM Gly-Gly, 2500mM NaCl, 8.03pH, 166.8 mS/cm

Controller set point = 15%B for 10x dilution to 25.1mS/cm

15/04/2005 / 20

10x Concentrate dilution at 120L/h

0 20 40 60 80 100 1200

102030405060708090

100

% B


10x Dilution

5x Dilution

CIR101 CIR102 AIR121pH FIR141 TIR101 SetMark

0

50

100

150

mS/cm

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

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

22.0

23.0

24.0

25.0

26.0

27.0

21.0

22.0

23.0

24.0

25.0

26.0

27.0

6.0 7.0 8.0 9.0 10.0 min

Feed

back

ena

bled

15/04/2005 / 21

Importance of in-line dilution for addressing demands of high titre batches.

Careful selection of buffers during early phase development can alleviate serious headaches at scale later

Select buffers (especially equilibration/wash) that can be prepared in concentrate form

Design capability for in-line dilution into chromatography and ultrafiltration rigs

Possible to obtain a 30-40% reduction in total buffer prep and hold requirements for equilibration buffers alone

15/04/2005 / 22


Throughput optimisation-Protein A capture

Recent advances in Protein A matrix designHigh throughput matricesHigh capacity matricesAlkali stable options

Explore relationship between titre increase and matrix selection

Examine impact of column diameter on process design and operation

15/04/2005 / 23

Effect of fermentation titre on column capacity

0 10 20 30 40 500

20

40

60

80

100

IgG loaded (mg/mL.matrix)

PnA Sepharose, 5g/L

Bre

akth

roug

h (C

/Co)

%

PnA Sepharose, 0.5g/L 10x increase in fermentation titre

83% increase in binding capacity under identical conditions

Despite increased binding capacity, compressibility limits attainable throughput and scale-up potential

0 100 200 300 400 500 600 7000

5

10

15

20

25

30

35

40

Pres

sure

Dro

p (p

si)

Linear velocity (cm/h)

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

ColumnDiameter

140cm

15/04/2005 / 24

Addressing chromatographic throughput

Incompressible matrices offer benefit of higher throughput.

Essential for high volume fermenters

Breakthrough curves generated at 450cm/h

(3x faster than Sepharose maximum )

23% increase in binding capacity at 5% C/Co under identical conditions.

0 10 20 30 40 500

20

40

60

80

100 MabSelect, 0.5g/L

Bre

akth

roug

h (C

/Co)

%

IgG loaded (mg/mL.matrix)

MabSelect, 5g/L

15/04/2005 / 25

Importance of matrix selection on process throughput at different titres

0.5g/L Titre 5g/L Titre

Rmp Protein ASepharose FF

MabSelect

ConclusionHigh throughput matrices can deliver 50% improvement in throughput at high fermentation titres

2000L Reactor

15/04/2005 / 26

Influence of column diameter on batch and campaign costs.

2000L reactor at 5g/LMabSelect for initial capture

“Small” PnA Column

“Large” PnA Column

No difference in campaigncost over 15 batches

15/04/2005 / 27

Factors to consider when selecting a column diameter

Range of fermentation titres across all products in portfolio (Flexibility)

A wide range in titre drives higher utilisation of narrower column diameters

Campaign length (number of batches)longer campaigns may favour large diameter columns

RiskBioburden contamination, operating close to re-use limits

Capital equipment requirementsmore chromatography skids, larger elution tanks)

Facility design, operation, logisticsfloor space-processing/storage, packing-unpacking

15/04/2005 / 28

Adressing capacity challenges with larger columns

Small diameter columns

Lower capital costsColumns and skids

Lower one-off batch costs

Increased flexibility across range of titres

Potential issues with high utilisation rates of equipment

MaintenanceColumn Re-packing

Large diameter columns

Higher capital costsColumns (stainless?)Skids

Extremely high first batch and replacement PnA costs

Large elution volumes per cycle

High exposure to risk through contamination (e.g. bio-burden) or other process failure

15/04/2005 / 29

Stay abreast of latest vendor offerings

1991 Protein A Seph 4FF

1996 rProtein A Seph 4FF

2000 rmp Protein A Seph 4FF

2001 MabSelect

2005….MabSelect SuRe, Xtra

Amersham Matrix launches.

E D A B C X MSs

Immunoglobulin binding domains

Gly29Ala mutation

Z domain

15/04/2005 / 30

Higher through, higher capacity, alkali stability

0 10 20 30 400

20

40

60

80

100 rmpPnA Seph 4 FF

Bre

akth

roug

h (C

/Co)

%

Antibody Loaded (mg/mL)

Mabselect

Prototype SuRe

Rmp Protein A 4 FFXK16, 15cm Ho, 150cm/h, 6min RT5% C/Co = 26g/LOp.DBC = 20.8g/L

MabSelectTC5, 20cm Ho, 500cm/h, 2.4min RT5% C/Co = 19g/LOp.DBC = 15.2/L

MabSelect SuReTC5, 20cm Ho, 500cm/h, 2.4min RT5% C/Co = 26g/LOp.DBC = 20.8g/L

15/04/2005 / 31

Strategies for Chromatography Process Development

Ensure process development teams understand manufacturing requirements:

High throughput per batch?Low batch costs?

Operate closer to 1% Breakthrough to minimise cycling requirements

Operate as close to matrix throughput limits as manufacturing capabilities permit (minimise residence time)

Employ latest matrix technologies

Ensure processes have detailed cost models to justify/defend process decisions

15/04/2005 / 32

Individual Reactor Volume established at 20,000L

LONZA BiologicsLSBO Facility

3x20,000L Reactors

Increasing to 4 Reactors in 2006

Purified through a Single Purification train

15/04/2005 / 33

Do ultra-large columns adequately meet high titre challenge?

LSBO Facility designed to handle high-titre batches

Protein A Capture in either a 1.4 or 2.0 m diameter Chromatography column.

Chromatographic capture of 20,000L at 5g/L feasible given facility design basis

15/04/2005 / 34



Early Phase fast track development may not allow sufficient time to optimise UF operations

Diafiltration conditions may be selected based onPrior experience with antibody class/pIPrior experience with particular cassette type/vendorManufacturing equipment capabilitiesAntibody stability

UF optimisation can substantially improve purification process performance

15/04/2005 / 35


TMP

Flux

-J

Low QMed QHigh Q

cTMP

Bulk Flow (Cb)

Boundary Layer

Gel Layer (Cg)Membrane

Increasing Concentration

Cb

Cg

Crossflow

eC

C gdf =

ln C CgFl

ux -

J

15/04/2005 / 36

Results from an Ultrafiltration optimisation

Membrane Un-Optimised Optimised

Gel Concentration (Cg) (g.L-1) 44.6 72.1

Optimum Process Conc (Cg/e) (g.L-1) 16.4 26.5

Flux at Cg/e (L.m-2.h-1) 31.1 59.3

Diafiltration Volume (L) 2,332 1,443

5 x Diafiltration Volume (L) 11,660 7,215

Concentration Time (h) 2 h 32 min 1 h 22 min

Diafiltration Time (h) 9h 22 min 3h 2 min

Total Process Time (h) 11 h 54 min 4 h 24 min

Un-optimised

Pin = 20 psig

Pout = 5 psig

Cross flow = 1x

Optimised

Pin = 35 psig

Pout = 12 psig

Cross flow = 1.33x

Membrane Area = 40 m2

Initial Volume = 7,300 L

Initial concentration = 5.24 g/L, concentrated to membrane Cg

15/04/2005 / 37

0 10 20 30 40 50 60 70 80 90 100 1100

2

4

6

8

10

12

14

Tota

l Pro

cess

Tim

e (h

)

Product Concentration at DF (g.L-1)

Omega Biomax Hydrosart Alpha 5 x DF Volume

0

2k

4k

6k

8k

10k

12k

14k

16k

18k

20k

Dia

filtra

te V

olum

e (L

)

Ultrafiltration OptimisationInfluence of Cassette type and vendor

KeyPolyethersulfone Vendor 1Polyethersulfone Vendor 2Regen. Cellulose Vendor 2Regen. Cellulose Vendor 3

Wide variation in optimised performance across different UF cassettes, especially at extremities of protein concentration

Model: 40kg batch

Membrane Area = 40 m2

Initial Volume = 7,300 L

Initial concentration = 5.24 g/L, concentrated to Cg, 5 diavolumes.

15/04/2005 / 38

Large Scale Ultrafiltration-80m2 capacity

15/04/2005 / 39


Optimisation of UF operations can deliver reduced processing times and considerable reduction in buffer volumes

Simple ultrafiltration optimisation can be performed in a single day with tremendous benefits

Avoid mid-range operating conditions-lead to un-optimised processes

Product stability at elevated concentrations should be assessed with appropriate stability studies

15/04/2005 / 40

Summary

Maximise utilisation of existing assets for as long as possible“Optimise” performance of unit operations early in development life cycle

Employ high throughput chromatography matrices

Select buffers with in-line dilution in mind

Strive for purification at elevated protein concentration wherever possible

However above strategy may have only a limited lifespan

Parallel track with investment into novel and conventional technologies

strategies for the purification of high titre, high volume

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