Downstream Processing

Product source and its impact

Setting project goals
Development of a process
The three stages in downstream
processing
Purification strategy
Applications

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Product source and its impact

The source of the product influences:

Product concentration
Extraction/solubilisation problems
Type and quantity of impurities
Product stability
Validation requirements

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Setting Project Goals

Regulatory requirements
Intended use of the product
Phase of development
Scale of manufacture
Economics
Facility
Contract manufacturing

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Development of a process
BIOREACTOR Chromatography Stages

———————————————————————————————

Cell Separation Cell Disruption

Clarified Culture Cell Debris

Medium Removal
Downstrem processing

Capture

Product Recovery
and Concentration

Intermediate
Column-Based Purification
Separation

Other Separation Polishing

Operation

Purified Bulk Drug

Substance

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 The three stages in downstream
processing
Purity

in g
ish
P ol
a t e • achieve final purity and safety
d i n • additional safety measure
m e ti o
t e r c a
In urif i • further removal of most proteins

e P • further removal of most nucleic acids

tu r • further removal of endotoxins

• further removal of viruses
ap • initial purification
C • stabilization
• clarification
• concentration
Step
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Purification Strategy

Resolution The optimal process must

be defined within the
context of competing
goals.

Speed
Loadability

Recovery
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Capture
z Initial purification of the target molecule from crude or
clarified source material
z Concentration and stabilization (e.g. removal of proteases)
z Removal of bulk impurities

Resolution

Speed
Loadability
Recovery
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Intermediate Purification
z Removal of same-class impurities
z Removal of "regulatory" impurities

Resolution

Speed
Loadability
Recovery
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Polishing
z Final removal of trace contaminants, e.g.
structural variants of the target protein
z Adjustment for storage
z Safety issues

Resolution

Speed
Loadability

Recovery 9/
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Three Phase Strategy -
Ranking of Chromatography
Techniques
Technique Capture Intermediate Polishing Considerations

GF Limited sample volume

Limited flow rate range

IEX

HIC

AC Protein ligands are

sensitive to harsh
cleaning conditions

RPC Use of organic solvents,

loss of biological activity

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Process purification of a recombinant
bacterial exotoxin
Unclarified E. coli lysate

STREAMLINE™ DEAE Capture

Phenyl Sepharose™ 6 Fast Flow (high sub)

Intermediate
Purification
SOURCE™ 30Q

SOURCE™ 15PHE Polishing

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Purification step 1:
One-step Capture with STREAMLINE™ DEAE
A 280 nm
Column: STREAMLINE 200, (20 cm diameter)
2.0
Medium: STREAMLINE DEAE, 4.7 litres

Heigh of expanded bed

Sample: 4.7 kg of cells were subjected to
osmotic shock and suspended in a
final volume of 180 litres 50 mM tris
buffer, pH 7.4, before application
100
1.0 onto the expanded bed.
80
Buffer A: 50 mM Tris buffer, pH 7.4
60
Buffer B: 50 mM Tris, 0.5 M sodium chloride,
40 pH 7.4
20 Flow rate: 400 cm/h during sample application
Sample Washing Elution
Buffer Buffer
and wash 100 cm/h during elution
100 200 260
Volume

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Purification step 2: Intermediate purification with
Phenyl Sepharose™ 6 Fast Flow (high sub.)
Column: BPG™ 200/500 (20 cm diameter) RPC analysis of pool obtained after purification
Medium: Phenyl Sepharose 6 Fast Flow (high with Phenyl Sepharose 6 Fast Flow (high sub.)
sub), 4.7 litres ammonium sulfate and
applied onto the column Analysis on µRPC C2/C18 with
Buffer A: 50 mM phosphate, 0.7 M ammonium SMART™ System
sulfate, pH 7.4
Buffer B: 20 mM phosphate, pH 7.4
C: distilled water
Flow rate: 120 cm/h A280nm
A 280 nm
0.50

0.40

0.30

0.20
water

0.10

Pool
Sample 0.00
Buffer A Buffer B Buffer C
0 2.0 4.0 6.0 8.0 Volume (ml)
6 12 18 24
Volume

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 S30 Exo 100

Purification step 3:
Intermediate purification with SOURCE™ 30Q
Column: FineLINE™ 100 (10 cm diameter) RPC analysis of pool obtained after
Medium: SOURCE 30Q, 375 ml purification with SOURCE 30Q
Sample: From the previous pool, diluted 1 to 3
with distilled water 1.5 litres/cycle
was applied
Buffer A: 20 mM phosphate, pH 7.4 A280nm

Buffer B: Buffer B + 1.0 M sodium chloride

Gradient: 0 to 50% B, 20 column volumes 0.30
Flow rate: 600 cm/h
A280nm
0.20
0.50

0.40
0.10
0.30

0.20
0.00
0.0 2.0 4.0 6.0 8.0 Volume (ml)
0.10
Pool
0.00
0.0 2.0 4.0 6.0 8.0 10.0 12.0
Volume (litres)

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Purification step 4:
Polishing with SOURCE™ 15PHE
Column: 35 MM I.D. X 100 MM RPC analysis of pool obtained
Medium: SOURCE 15PHE
Sample: from the previous step, adjusted to
1.0 M ammonium sulfate, A280nm
0.5 litres/cycle was applied
0.40
Buffer A: 1.0 M ammonium sulfate, 50 mM
phosphate, pH 7.4
Buffer B: 50 mM phosphate 0.30
Gradient: 0 to 45% B, 15 column volumes
Flow rate: 200 cm/h 0.20
A280nm

0.10
0.15

0.00
0.10

0.0 2.0 4.0 6.0 8.0 Volume (ml)

0.05

Pool

0.00
0 20 40 60 Time (min)

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Conclusions
• The use of novel techniques and chromatography media for Capture,
Intermediate Purification and Polishing enabled the production of a highly
purified exotoxin A from crude cell homogenate using only four
chromatographic steps.

• The use of STREAMLINE™ for removal of cell debris and particulate

matter and Capture of target protein eliminated the need for traditional
clarification procedures such as high speed centrifugation followed by
microfiltration. It not only eliminated the clarification step, but also
produced a concentrated and partly purified product, ready for
Intermediate Purification.
• The power of combining established techniques such as HIC and IEX with
modern high performance media allows the use of high flow rates, facilitates
process development and shortens process time.

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Our main Downstream application areas

Recombinant proteins & peptides

MAbs
Biologicals from natural sources
Synthetic oligonucleotides & peptides
Plasmids

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Purification of recombinant α-amylase
from E. coli
Column: INdEX™ 70 (70 mm i.d.)
Adsorbent: Q Sepharose™ XL, 385 mL bed volumne
Sample: Recombinant α-amylase produced in E. coli,
Lane 1: -
homogenized, 2.2 L diluted in distilled water to Lane 2: LMW markers
15.4 L, 7.2 mS/cm, 10 mM CaCl2, centrifuged Lane 3: Starting material
Buffer A: 20 mM Tris-HCl, pH 8, 10 mM CaCl2 Lane 4: Flow through
Buffer B: 20 mM Tris-HCl, pH 8, 1 M NaCl, 10 mM CaCl2 Lane 5: 1st peak
Flow rate: 300 cm/h, 12 L/h
Gradient: 20 bed volumes 0-1 M NaCl
(containing α-amylase)
Eluate: 1.48 L, 3.8 bed volumes Lane 6: 2nd peak
Spec. act. Lane 7: LMW markers
α-amylase 6420 U/L

A280 Cond

0 5.0 10.0 15.0 20.0 Volume (l) 12 3 4 5 6 7

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Scale up of the purification of a humanized IgG4
antibody from myeloma cell culture broth by
expanded bed affinity adsorption
A280 a) Column: STREAMLINE™ 25 (25 mm i.d.)
Adsorbent: STREAMLINE rProtein A
Feed: 1.5 litres of myeloma cell culture
Buffer A: 50 mM glycine, 250 mM NaCl, pH 8
Buffer B: 100 mM glycine pH 3.0
Flow velocity: 300 cm/h at equilibration, feed
80 160 application and wash 100 cm/h at
Sample loading Washing Washing Elution elution and CIP
(expanded mode) (packed mode) Time (min)

A280

b) Column: STREAMLINE 200 (200 mm i.d.)

Adsorbent: STREAMLINE rProtein A
Feed: 93 litres of myeloma cell culture
Buffer A: 50 mM glycine, 250 mM NaCl, pH 8
Buffer B: 100 mM glycine pH 3.0
Flow velocity: 300 cm/h at equilibration, feed
application and wash 100 cm/h at
80 160
Sample loading Washing Washing Elution
elution and CIP
(expanded mode) (packed mode) Time (min)

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Purification of a humanized IgG4 antibody from
myeloma cell culture broth by expanded bed
affinity adsorption
STREAMLINE™ 25 STREAMLINE 200
run 1 run 2
Adsorbent (L) 0.075 0.075 4.7
Feed
Volume (L) 1.5 1.5 93
Conc. IgG4 (mg/ml) 0.407 0.407 0.407
Amount IgG4 (mg) 611 611 37900
Eluate
Volume (L) 0.0912 0.0912 6.134
Conc. IgG4 (mg/ml) 7.41 7.46 7.68
Amount IgG4 (mg) 676 677 47109
BSA (ng/mg IgG4) 8.6 25 10
Transferrin (ng/mg IgG4) <0.2 <0.4 <0.4
DNA (pg/mg IgG4) N/A N/A <0.26
Yield (%) 111 111 124

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Purification of Plasma Proteins
Whole blood

Platelets Red cells

Plasma for fractionation

F VIII F IX Albumin IgG

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Procedure for production of
Immunoglobulin
fresh frozen plasma without coagulation
factors or outdated plasma

Sephadex™ G-25 C
euglobin ppt I and pH
adjustment
DEAE Sepharose™ Fast Flow
pH and I
adjustment
Q Sepharose Fast Flow

CM Sepharose Fast Flow

ultrafiltration
virus inactivation
solvent/detergent
CM Sepharose Fast Flow
ultrafiltration
formulation
IgG powder 5.0 g/L sterile filtration
of plasma lyophilization 22 /
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Purification of oligonucleotides on
SOURCE™ 15Q
An analytical chromatogram obtained for the crude starting material,
analysed by ion exchange chromatography

Column: RESOURCE™ Q, 1 ml
mAU %B
Sample: 25µL
100
Flow: 0.5 mL/min
600 80 Eluent A: 10 mM NaOH, 1.0 M NaCl
Eluent B: 10 mM NaOH, 3.0 M NaCl
60 Gradient: 0-45% B, 17.5 column vol.
400

40
System: ÄKTA™purifier
200
Detection: 260 nm
20

0 0
0.0 5.0 10.0 15.0 20.0 Volume (mL)

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Purification of oligonucleotides on
SOURCE™ 15Q
Column: FineLINE™ Pilot 35 Column: Customized FineLINE 800
Flow: 8 mL/min: 50 cm/h Flow: 250 L/min; 50 cm/h
System: ÄKTA™explorer System: BioProcess™ Engineering 8 mm, 10 bar
Detection: 280 nm Detection: 280 nm

mAU mS/cm AU mS/cm

2500 150 150

1.5
2000

1500 100 100

1.0

1000
0 0.5 0
500

0 0.0
0 200 400 600 800 Volume (L) 100 200 300 400 500 L

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Purification of oligonucleotides on
SOURCE™ 15Q
An analytical chromatogram of obtained for the final product

Column: RESOURCE™ Q, 1 ml
Sample: 25 µl
mAU %B
100 Flow: 0.5 mL/min
Eluent A: 10 mM NaOH, 1.0 M NaCl
80 Eluent B: 10 mM NaOH, 3.0 M NaCl
1000 Gradient: 0-45% B, 17.5 column vol.
60
System: ÄKTA™purifier
40
Detection: 260 nm
500

20

0 0
0.0 5.0 10.0 15.0 20.0 Volume (ml)

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Purification of Plasmids
Cell paste Resuspension in buffer
Alkaline lysis
Neutralisation

Addition of CaCl2 Centrifugation

Filtration - glass wool
Filtration 0.45 µm
Capture - Q Sepharose™ XL

Polishing - SOURCE™ 15Q

Plasmids for gene therapy

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

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Purification of Plasmids
Polishing on SOURCE™ 15Q

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Purification of Plasmids
Analysis of plasmid fractions from Capture and Polishing

Q Sepharose XL SOURCE 15Q

Bed volume 10 ml Bed volume 6 ml
Amount of plasmid applied (mg) 6.2 4.6
Analysis of plasmid peak
Total nucleic acid (mg) 5.4 3.8
Plasmid amount (mg) 4.8 3.9
Plasmid (% nucleic acid) 90 99.4
RNA (% nucleic acid) not detected
Chromosomal DNA (% nucleic acid) 2 0.6
Supercoiled plasmid (% plasmid) 93 100
Plasmid yield % per step 78 85
Yield % (total plasmid) 78 66

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