02 Downstream Process

8/6/2019 02 Downstream Process

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1 /GE /

Downstream Processing

Product source and its impact

Setting project goalsDevelopment of a process

The three stages in downstreamprocessingPurification strategy

Applications



2 /GE /

Product source and its impact

Product concentrationExtraction/solubilisation problems

Type and quantity of impuritiesProduct stability

Validation requirements

The source of the product influences:



3 /GE /

Setting Project Goals

Regulatory requirements

Intended use of the productPhase of development

Scale of manufactureEconomics

FacilityContract manufacturing



4 /GE /

Development of a processBIOREACTORBIOREACTOR

Cell SeparationCell Separation Cell DisruptionCell Disruption

CaptureCapture

IntermediatePurification


PolishingPolishing

Chromatography Stages

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

D o w n s t r e m p r o c e s s i n g

Product Recovery

and Concentration

Product Recovery

and Concentration

Column-BasedSeparation

Column-BasedSeparation

Other SeparationOperation

Other SeparationOperation

Purified Bulk DrugSubstance

Purified Bulk DrugSubstance

Cell DebrisCell DebrisRemovalRemoval

Clarified CultureClarified CultureMediumMedium



5 /GE /

The three stages in downstream

processing

Step

Purity

• further removal of most proteins• further removal of most nucleic acids• further removal of endotoxins

• further removal of viruses• initial purification• stabilization• clarification• concentration

• achieve final purity and safety• additional safety measure

P o l i s h i

n g

C a p t u r e

I n t e r m e d i

a t e

P u r i f i

c a t i o n



6 /GE /

Purification Strategy

RecoveryRecovery

ResolutionResolution

SpeedSpeed LoadabilityLoadability

The optimal process must

be defined within thecontext of competinggoals.



8 /GE /

Intermediate PurificationRemoval of same-class impuritiesRemoval of "regulatory" impurities

Recovery


SpeedLoadabilityLoadability



9 /GE /

PolishingFinal removal of trace contaminants, e.g.structural variants of the target proteinAdjustment for storageSafety issues

Recovery

SpeedLoadability




10 /GE /

Three Phase Strategy -Ranking of ChromatographyTechniques

Considerations

Limited sample volumeLimited flow rate range

Protein ligands aresensitive to harshcleaning conditions

Use of organic solvents,loss of biological activity

Technique Capture Intermediate Polishing

GF

IEX

HIC

AC

RPC



11 /GE /

Process purification of a recombinant

bacterial exotoxin


E. coli lysateE. coli lysate

STREAMLINE™ DEAESTREAMLINE™ DEAE

Phenyl Sepharose ™ 6 Fast Flow (high sub)Phenyl Sepharose ™ 6 Fast Flow (high sub)

SOURCE™ 30QSOURCE™ 30Q

SOURCE™ 15PHESOURCE™ 15PHE

Capture

Polishing

Unclarified



12 /GE /

Purification step 1:

One-step Capture with STREAMLINE™ DEAEColumn: STREAMLINE 200, (20 cm diameter)

Medium: STREAMLINE DEAE, 4.7 litres

Sample: 4.7 kg of cells were subjected toosmotic shock and suspended in afinal volume of 180 litres 50 mM trisbuffer, pH 7.4, before applicationonto the expanded bed.

Buffer A: 50 mM Tris buffer, pH 7.4

Buffer B: 50 mM Tris, 0.5 M sodium chloride,pH 7.4

Flow rate: 400 cm/h during sample applicationand wash 100 cm/h during elution

100 200 260Volume

2.0

1.0

A 280 nm

20

40

60

80

100

H e

i g h o

f e x p a n

d e

d b e

d

Sample ElutionBuffer Buffer

Washing



13 /GE /

Purification step 2: Intermediate purification with

Phenyl Sepharose™ 6 Fast Flow (high sub.)RPC analysis of pool obtained after purificationwith Phenyl Sepharose 6 Fast Flow (high sub.)

Analysis on µRPC C2/C18 withSMART™ System

Column: BPG™ 200/500 (20 cm diameter)Medium: Phenyl Sepharose 6 Fast Flow (high

sub), 4.7 litres ammonium sulfate andapplied onto the column

Buffer A: 50 mM phosphate, 0.7 M ammoniumsulfate, pH 7.4

Buffer B: 20 mM phosphate, pH 7.4C: distilled water

Flow rate: 120 cm/h

6 12Volume

A 280 nm

SampleBuffer A Buffer B

18 24Buffer C

w a

t e r

Pool

A280nm

0.50

0.40

0.30

0.20

0.10

0.000 2.0 4.0 6.0 8.0 Volume (ml)

S30 E 100



14 /GE /


Intermediate purification with SOURCE™ 30QRPC analysis of pool obtained afterpurification with SOURCE 30Q

Column: FineLINE™ 100 (10 cm diameter)Medium: SOURCE 30Q, 375 mlSample: From the previous pool, diluted 1 to 3

with distilled water 1.5 litres/cyclewas appliedBuffer A: 20 mM phosphate, pH 7.4Buffer B: Buffer B + 1.0 M sodium chlorideGradient: 0 to 50% B, 20 column volumesFlow rate: 600 cm/h

S30 Exo 100

A 280nm

0.50

0.40

0.30

0.20

0.10

0.00

0.0 2.0 4.0 6.0 8.0 10.0 12.0Volume (litres)

Pool

A280nm

0.30

0.20

0.10

0.000.0 2.0 4.0 6.0 8.0 Volume (ml)



15 /GE /


Polishing with SOURCE™ 15PHEColumn: 35 MM I.D. X 100 MMMedium: SOURCE 15PHESample: from the previous step, adjusted to

1.0 M ammonium sulfate,0.5 litres/cycle was appliedBuffer A: 1.0 M ammonium sulfate, 50 mM

phosphate, pH 7.4Buffer B: 50 mM phosphateGradient: 0 to 45% B, 15 column volumes

Flow rate: 200 cm/h

RPC analysis of pool obtained

A 280nm

0.15

0.10

0.05

0.000 20 40 60 Time (min)

Pool

Volume (ml)

A280nm

0.40

0.30

0.20

0.10

0.00

0.0 2.0 4.0 6.0 8.0



16 /GE /

Conclusions• The use of novel techniques and chromatography media for Capture,

Intermediate Purification and Polishing enabled the production of a highlypurified exotoxin A from crude cell homogenate using only fourchromatographic steps.

• The use of STREAMLINE™ for removal of cell debris and particulatematter and Capture of target protein eliminated the need for traditional

clarification procedures such as high speed centrifugation followed bymicrofiltration. It not only eliminated the clarification step, but alsoproduced a concentrated and partly purified product, ready forIntermediate Purification.

• The power of combining established techniques such as HIC and IEX withmodern high performance media allows the use of high flow rates, facilitatesprocess development and shortens process time.



17 /GE /

Our main Downstream application areas

Recombinant proteins & peptidesMAbsBiologicals from natural sourcesSynthetic oligonucleotides & peptidesPlasmids



18 /GE /

Purification of recombinant α -amylase

from E. coli

A280 Cond

0 5.0 Volume (l)10.0 15.0 20.0

Column: INdEX™ 70 (70 mm i.d.)Adsorbent: Q Sepharose™ XL, 385 mL bed volumneSample: Recombinant α -amylase produced in E. coli ,

homogenized, 2.2 L diluted in distilled water to15.4 L, 7.2 mS/cm, 10 mM CaCl 2, centrifuged

Buffer A: 20 mM Tris-HCl, pH 8, 10 mM CaCl 2

Buffer B: 20 mM Tris-HCl, pH 8, 1 M NaCl, 10 mM CaCl 2Flow rate: 300 cm/h, 12 L/hGradient: 20 bed volumes 0-1 M NaClEluate: 1.48 L, 3.8 bed volumesSpec. act.α -amylase 6420 U/L

1 2 3 4 5 6 7

Lane 1: -Lane 2: LMW markersLane 3: Starting materialLane 4: Flow throughLane 5: 1st peak(containingα -amylase)Lane 6: 2nd peakLane 7: LMW markers

l f h f f h d



19 /GE /

Scale up of the purification of a humanized IgG 4antibody from myeloma cell culture broth byexpanded bed affinity adsorption

A280

80 160Washing(packed mode)

Washing(expanded mode) Time (min)

ElutionSample loading

A280

80 160Washing(packed mode)

Washing(expanded mode) Time (min)

ElutionSample loading

Column: STREAMLINE™ 25 (25 mm i.d.)Adsorbent: STREAMLINE rProtein AFeed: 1.5 litres of myeloma cell cultureBuffer A: 50 mM glycine, 250 mM NaCl, pH 8Buffer B: 100 mM glycine pH 3.0Flow velocity: 300 cm/h at equilibration, feed

application and wash 100 cm/h atelution and CIP

a)

Column: STREAMLINE 200 (200 mm i.d.)Adsorbent: STREAMLINE rProtein AFeed: 93 litres of myeloma cell cultureBuffer A: 50 mM glycine, 250 mM NaCl, pH 8Buffer B: 100 mM glycine pH 3.0Flow velocity: 300 cm/h at equilibration, feed

application and wash 100 cm/h atelution and CIP

b)

P ifi i f h i d I G ib d f



20 /GE /

Purification of a humanized IgG 4 antibody frommyeloma cell culture broth by expanded bedaffinity adsorption

Adsorbent (L) 0.075 0.075 4.7Feed

Volume (L) 1.5 1.5 93Conc. IgG4 (mg/ml) 0.407 0.407 0.407Amount IgG

4(mg) 611 611 37900

EluateVolume (L) 0.0912 0.0912 6.134Conc. IgG4 (mg/ml) 7.41 7.46 7.68Amount IgG4 (mg) 676 677 47109

BSA (ng/mg IgG4) 8.6 25 10Transferrin (ng/mg IgG4) <0.2 <0.4 <0.4DNA (pg/mg IgG4) N/A N/A <0.26

Yield (%) 111 111 124

STREAMLINE™ 25STREAMLINE™ 25 STREAMLINE 200STREAMLINE 200run 1run 1 run 2run 2



22 /GE /

Sephadex™ G-25 CSephadex™ G-25 C

DEAE Sepharose™ Fast FlowDEAE Sepharose™ Fast Flow

Q Sepharose Fast FlowQ Sepharose Fast Flow

CM Sepharose Fast FlowCM Sepharose Fast Flow

CM Sepharose Fast FlowCM Sepharose Fast Flow

Procedure for production of

Immunoglobulinfresh frozen plasma without coagulationfactors or outdated plasma

euglobin ppt I and pHadjustment

pH and Iadjustment

ultrafiltrationvirus inactivationsolvent/detergent

ultrafiltrationformulation

sterile filtrationlyophilizationIgG powder 5.0 g/Lof plasma



23 /GE /

Purification of oligonucleotides on

SOURCE™ 15QAn analytical chromatogram obtained for the crude starting material,analysed by ion exchange chromatography

mAU

600

400

200

0

0.0 5.0 10.0 15.0 20.0 Volume (mL)

%B

100

80

60

40

20

0

Column: RESOURCE™ Q, 1 mlSample: 25µLFlow: 0.5 mL/minEluent A: 10 mM NaOH, 1.0 M NaClEluent B: 10 mM NaOH, 3.0 M NaClGradient: 0-45% B, 17.5 column vol.System: ÄKTA™purifier Detection: 260 nm



24 /GE /


SOURCE™ 15Q

mAU

2500

2000

1500

1000

500

00 200 400 600 800 Volume (L)

mS/cm

150

100

0

Column: FineLINE™ Pilot 35Flow: 8 mL/min: 50 cm/hSystem: ÄKTA™explorer

Detection: 280 nm

AU

1.5

1.0

0.5

0.0100 200 300 400 500 L

mS/cm

150

100

0

Column: Customized FineLINE 800Flow: 250 L/min; 50 cm/hSystem: BioProcess™ Engineering 8 mm, 10 bar

Detection: 280 nm



25 /GE /


SOURCE™ 15QAn analytical chromatogram of obtained for the final product

mAU

1000

500

00.0 5.0 10.0 15.0 20.0 Volume (ml)

%B100

80

60

40

20

0

Column: RESOURCE™ Q, 1 mlSample: 25 µlFlow: 0.5 mL/minEluent A: 10 mM NaOH, 1.0 M NaCl

Eluent B: 10 mM NaOH, 3.0 M NaClGradient: 0-45% B, 17.5 column vol.System: ÄKTA™purifier Detection: 260 nm



26 /GE /

Purification of PlasmidsCell paste

Addition of CaCl2

Capture - Q Sepharose™ XL

Polishing - SOURCE™ 15Q

Plasmids for gene therapy

Resuspension in bufferAlkaline lysisNeutralisation

CentrifugationFiltration - glass woolFiltration 0.45 µm



27 /GE /

Purification step 1:Capture of plasmid on Q Sepharose™ XL



28 /GE /

Purification of PlasmidsPolishing on SOURCE™ 15Q



29 /GE /

Purification of PlasmidsAnalysis of plasmid fractions from Capture and Polishing

Amount of plasmid applied (mg) 6.2 4.6Analysis of plasmid peakTotal nucleic acid (mg) 5.4 3.8

Plasmid amount (mg) 4.8 3.9Plasmid (% nucleic acid) 90 99.4RNA (% nucleic acid) not detectedChromosomal DNA (% nucleic acid) 2 0.6Supercoiled plasmid (% plasmid) 93 100Plasmid yield % per step 78 85Yield % (total plasmid) 78 66

Q Sepharose XLBed volume 10 mlQ Sepharose XL

Bed volume 10 mlSOURCE 15Q

Bed volume 6 mlSOURCE 15Q

Bed volume 6 ml

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