Scale-up with SOURCE 15PHE
Scale-Up at the Polishing Stage of
Downstream Processes
Background
Polishing of a recombinant Pseudomonas aeruginosa exotoxin A, for the preparation of polysaccharide conjugated
vaccines, was performed on SOURCE 15PHE. Previous purification involved STREAMLINE™ DEAE, Phenyl
Sepharose Fast Flow (high sub) and SOURCE 30Q.
Column:
Medium:
Sample:
FineLINE 100 (100 mm i.d.)
SOURCE 30Q, 375 ml
From the previous pool, diluted 1 to 3 with distilled
water, and 1.5 litres/cycle was applied
20 mM phosphate, pH 7.4
Buffer B + 1.0 M sodium chloride
0–50% B, 20 column volumes
600 cm /h
Buffer A:
Buffer B:
Gradient:
Flow:
Column:
Medium:
Sample:
FineLINE Pilot 35 (35 mm i.d. x 100 mm)
SOURCE 15PHE, 96 ml
From the previous step, adjusted to
1.0 M ammonium sulphate, and 0.5 litres/cycle
was applied
1.0 M ammonium sulphate,
50 mM phosphate, pH 7.4
50 mM phosphate, pH 7.4
0 to 45% B, 15 column volumes
200 cm/h
Buffer A:
Buffer B:
Gradient:
Flow:
A 280nm
Åke Danielsson, Ingemar Daniels, Makonnen Belew, Bo Forsberg and Hans J. Johansson,
Amersham Biosciences, SE-751 84 Uppsala, Sweden
Purpose
To achieve consistent scale-up at the polishing stage of a downstream process, with different
chromatography techniques.
Introduction
The main aim at the final, polishing stage of a downstream process for a biopharmaceutical is to remove
trace contaminants such as structural variants of the product. To achieve consistent scale-up from the lab
bench to the production hall of these difficult separations can be a major bottleneck in process development.
A 280nm
0.50
0.15
0.40
0.30
We have used SOURCE™ chromatography media based on monosized 15 and 30 µm divinylbenzene/polystyrene beads for this task (Fig. 1). Using these media, the required high resolution was achieved at low or
moderate operating back-pressures (Fig. 2-3).
0.10
0.20
Scaled-up separations with SOURCE media for RPC, HIC and IEX are shown in Figures 4-9. Scale-up was
facilitated by the use of a uniform control platform, UNICORN™, which has a common user interface
for all scales of operation.
0.05
0.10
Pool
Pool
0.00
0.0
0.00
2.0
4.0
6.0
8.0
10.0
12.0
Volume (litres)
Fig 7. Purification of r-exotoxin A on SOURCE 30Q.
Recovery of exotoxin A was 92% (by immunodiffusion).
0
20
40
60
Time (min)
Fig 8. Polishing of r-exotoxin A on SOURCE 15PHE.
A 280nm
Low back-pressure
A 280nm
0.40
a
b
0.30
Back-pressure in bar
Back-pressure in bar
25
40
Bed height
0.30
20
0.20
0.20
15 cm
10 cm
5 cm
3 cm
30
25
15
0.10
0.10
35
SOURCE 30
Other 1
Other 2
20
10
0.00
15
0.00
0.0
2.0
4.0
6.0
8.0
Volume (ml)
0.0
Fig 9. HPLC analysis of r-exotoxin A (a) before and (b) after polishing on SOURCE 15PHE.
2.0
4.0
6.0
10
8.0
Volume (ml)
5
5
0
0
0
Summary
Polishing separations on SOURCE 15 and SOURCE 30 media were consistently scaled-up from
laboratory scale to pilot, and production scale.
Monosized SOURCE 30 media gave back-pressures below 10 bar at flow rates up to 1800 cm/h
(10 cm bed height, water, room temperature).
Consistent resolution was obtained at 300 cm/h and 1000 cm/h when 100 mm i.d. and 800 mm
i.d. FineLINE production columns packed with SOURCE 30S were compared.
RAK design AB.
SOURCE,UNICORN, Sepharose, RESOURCE, FineLINE, BioPilot, BioProcess and STREAMLINE are trademarks of Amersham
Pharmacia Biotech Limited or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc. Pharmacia and Drop
Design are trademarks of Pharmacia & Upjohn. Amersham Pharmacia Biotech AB Björkgatan 30, SE-751 84 Uppsala,
Sweden. Amersham Pharmacia Biotech UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England.
Amersham Pharmacia Biotech Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855 USA. Amersham Pharmacia
Biotech Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany. Amersham Pharmacia Biotech K.K. Sanken Building,
3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan. All goods and services are sold subject to the terms and
conditions of sale of the company within the Amersham Pharmacia Biotech group that supplies them. A copy of these terms
and conditions is available on request. © Amersham Pharmacia Biotech AB 2000 – All rights reserved.
Fig 1. Electron micrograph of SOURCE 30
beads. Note the uniform size and absence
of fines, fragments, and broken beads.
18-1124-77
Edition AB
400
800
1200
1600
2000
Linear flow in cm/h
Fig 2. Pressure/flow rate characteristics of
SOURCE 30. The monosized SOURCE 30
matrix was compared with polysized matrices
on the market. (Other 1 is a nominally 50 µm
diameter particle; Other 2 is a nominally
35 µm particle. Both were handled according
to the manufacturers recommendation.)
0
200
400
600
800
1000
Linear flow in cm/h
Fig 3. Back-pressure of SOURCE 15 in
large-scale columns. The experiments
were performed in a FineLINE™ 100
column (100 mm i.d.).
Scale-up with SOURCE 15RPC
Scale-up with SOURCE 30S
Table 1 rh-Epidermal growth factor (rh-EGF) variants expressed
in yeast.
Table 2 Purification process for rh-EGF.
1–52 (with N-terminal Ser), major form
Yeast cell culture supernatant
1–52 with oxidized Met
1–51 (with N-terminal Asp)
Column:
a) FineLINE Pilot 35, 35 mm i.d. x 109 mm (105 ml)
b) FineLINE 100, 100 mm i.d. x 100 mm (0.78 l)
c) FineLINE 800, 800 mm i.d. x 100 mm (50 l)
SOURCE 30S
Ribonuclease A, cytochrome C, and lysozyme,
all from Sigma (3.75:1:1)
0.32 mg total protein/ml media
20 mM sodium phosphate, pH 6.8
20 mM sodium phosphate + 0.4 M NaCl, pH 6.8
300 cm/h a) 48 ml/min, b) 0.39 l/min, c) 25 l/min
0–100% B in 20 column volumes
a) BioPilot™ b) and c) BioProcess™ Engineering
systems
Medium:
Sample:
Centrifugation or 5 um filtration
1–51 with oxidized Met
All variants have full activity in EGF receptor binding and
mitogen assays.
Phenyl Sepharose™ Fast Flow (high sub)
All variants have correct disulphide bond locations.
Q Sepharose High Performance
Sample load:
Buffer A:
Buffer B:
Flow:
Gradient:
System:
(Reference: Nascimento et al., Biochemistry 27, 797-802
(1988))
Sample:
Mixture of ribonuclease A, cytochrome C,
and lysozyme
0.32 mg/ml resin
20 mM sodium phosphate, pH 6.8
A + 0.4 M NaCl
a) and c) 300 cm/h
b) and d)1000 cm/h
Load:
Eluent A:
Eluent B:
Flow:
FineLINE 100
300 cm/h
A 280 nm
0.080
a
0.060
SOURCE 15RPC
105 ml column
A 280 nm
0.040
0.040
a
0.020
0.030
0.000
a)
Column: RESOURCE™ RPC, 3 ml (6.4x100 mm)
Sample: 2.14 ml EGF pool
Flow:
1.6 ml/min (300 cm/h)
c), d)
HPLC analysis of rh-EGF before (c) and after (d) separation with
SOURCE 15RPC
20.0
15.0
10.0
0.020
Volume (l)
FineLINE 100
1000 cm/h
A 280 nm
0.010
b
0.050
0.000
A 280 nm
%B
100
1000
1500
0.040
2500
2000
Volume (ml)
c
0.030
a
0.060
0.020
80
0.010
780 ml column
A 280 nm
60
0.040
b
0.020
0.060
20
0.000
A 280 nm
0.040
50.0
0.020
40.0
20.0
15.0
10.0
0.080
40
Volume (l)
FineLINE 800
300 cm/h
c
0
0
40
20
60
80
Time (min)
d
A 280 nm
%B
30.0
0.000
100
0.10
20.0
15.0
10.0
b
Volume (l)
20.0
80
0.08
10.0
60
0.06
50 l column
A 280 nm
600
800
1000
1200
c
0.04
40
0.02
20
0.00
0
50.0
A 280 nm
40.0
b)
Column:
Sample:
Flow:
Recovery:
20
40
60
30.0
20.0
Time (min)
SOURCE 15RPC, FineLINE Pilot 35 (35x100 mm)
62.5 ml EGF pool
1.6 ml/min (300 cm/h)
92% of applied protein in EGF pool
Fig 4. Scale-up of rh-EGF polishing on SOURCE 15RPC.
Eluent A: 0.05% TFA, 5% acetonitrile in water, Eluent B: 0.05% TFA, 80% acetonitrile in water, Gradient: 0–100%B in 40
column volumes.
FineLINE 800
1000 cm/h
d
30.0
0
1400
Volume (l)
20.0
10.0
600
800
1000
1200
1400
Volume (l)
10.0
600
Fig 5. Scale-up from FineLINE Pilot 35 column via FineLINE
100 column (7x) to FineLINE 800 custom-designed column
(64x). Total scale-up factor was 476-fold.
800
1000
1200
1400
Volume (l)
Fig 6. Separations on SOURCE 30S in production columns, at
different flow rates.
a) and b) FineLINE 100 column (100 mm x 100 mm; 0.79 liter)
c) and d) FineLINE 800 column (800 mm x 100 mm; 50 liter)
Scale-up with SOURCE 15RPC
Scale-up with SOURCE 30S
Table 1 rh-Epidermal growth factor (rh-EGF) variants expressed
in yeast.
Table 2 Purification process for rh-EGF.
1–52 (with N-terminal Ser), major form
Yeast cell culture supernatant
1–52 with oxidized Met
1–51 (with N-terminal Asp)
Column:
a) FineLINE Pilot 35, 35 mm i.d. x 109 mm (105 ml)
b) FineLINE 100, 100 mm i.d. x 100 mm (0.78 l)
c) FineLINE 800, 800 mm i.d. x 100 mm (50 l)
SOURCE 30S
Ribonuclease A, cytochrome C, and lysozyme,
all from Sigma (3.75:1:1)
0.32 mg total protein/ml media
20 mM sodium phosphate, pH 6.8
20 mM sodium phosphate + 0.4 M NaCl, pH 6.8
300 cm/h a) 48 ml/min, b) 0.39 l/min, c) 25 l/min
0–100% B in 20 column volumes
a) BioPilot™ b) and c) BioProcess™ Engineering
systems
Medium:
Sample:
Centrifugation or 5 um filtration
1–51 with oxidized Met
All variants have full activity in EGF receptor binding and
mitogen assays.
Phenyl Sepharose™ Fast Flow (high sub)
All variants have correct disulphide bond locations.
Q Sepharose High Performance
Sample load:
Buffer A:
Buffer B:
Flow:
Gradient:
System:
(Reference: Nascimento et al., Biochemistry 27, 797-802
(1988))
Sample:
Mixture of ribonuclease A, cytochrome C,
and lysozyme
0.32 mg/ml resin
20 mM sodium phosphate, pH 6.8
A + 0.4 M NaCl
a) and c) 300 cm/h
b) and d)1000 cm/h
Load:
Eluent A:
Eluent B:
Flow:
FineLINE 100
300 cm/h
A 280 nm
0.080
a
0.060
SOURCE 15RPC
105 ml column
A 280 nm
0.040
0.040
a
0.020
0.030
0.000
a)
Column: RESOURCE™ RPC, 3 ml (6.4x100 mm)
Sample: 2.14 ml EGF pool
Flow:
1.6 ml/min (300 cm/h)
c), d)
HPLC analysis of rh-EGF before (c) and after (d) separation with
SOURCE 15RPC
20.0
15.0
10.0
0.020
Volume (l)
FineLINE 100
1000 cm/h
A 280 nm
0.010
b
0.050
0.000
A 280 nm
%B
100
1000
1500
0.040
2500
2000
Volume (ml)
c
0.030
a
0.060
0.020
80
0.010
780 ml column
A 280 nm
60
0.040
b
0.020
0.060
20
0.000
A 280 nm
0.040
50.0
0.020
40.0
20.0
15.0
10.0
0.080
40
Volume (l)
FineLINE 800
300 cm/h
c
0
0
40
20
60
80
Time (min)
d
A 280 nm
%B
30.0
0.000
100
0.10
20.0
15.0
10.0
b
Volume (l)
20.0
80
0.08
10.0
60
0.06
50 l column
A 280 nm
600
800
1000
1200
c
0.04
40
0.02
20
0.00
0
50.0
A 280 nm
40.0
b)
Column:
Sample:
Flow:
Recovery:
20
40
60
30.0
20.0
Time (min)
SOURCE 15RPC, FineLINE Pilot 35 (35x100 mm)
62.5 ml EGF pool
1.6 ml/min (300 cm/h)
92% of applied protein in EGF pool
Fig 4. Scale-up of rh-EGF polishing on SOURCE 15RPC.
Eluent A: 0.05% TFA, 5% acetonitrile in water, Eluent B: 0.05% TFA, 80% acetonitrile in water, Gradient: 0–100%B in 40
column volumes.
FineLINE 800
1000 cm/h
d
30.0
0
1400
Volume (l)
20.0
10.0
600
800
1000
1200
1400
Volume (l)
10.0
600
Fig 5. Scale-up from FineLINE Pilot 35 column via FineLINE
100 column (7x) to FineLINE 800 custom-designed column
(64x). Total scale-up factor was 476-fold.
800
1000
1200
1400
Volume (l)
Fig 6. Separations on SOURCE 30S in production columns, at
different flow rates.
a) and b) FineLINE 100 column (100 mm x 100 mm; 0.79 liter)
c) and d) FineLINE 800 column (800 mm x 100 mm; 50 liter)
Scale-up with SOURCE 15PHE
Scale-Up at the Polishing Stage of
Downstream Processes
Background
Polishing of a recombinant Pseudomonas aeruginosa exotoxin A, for the preparation of polysaccharide conjugated
vaccines, was performed on SOURCE 15PHE. Previous purification involved STREAMLINE™ DEAE, Phenyl
Sepharose Fast Flow (high sub) and SOURCE 30Q.
Column:
Medium:
Sample:
FineLINE 100 (100 mm i.d.)
SOURCE 30Q, 375 ml
From the previous pool, diluted 1 to 3 with distilled
water, and 1.5 litres/cycle was applied
20 mM phosphate, pH 7.4
Buffer B + 1.0 M sodium chloride
0–50% B, 20 column volumes
600 cm /h
Buffer A:
Buffer B:
Gradient:
Flow:
Column:
Medium:
Sample:
FineLINE Pilot 35 (35 mm i.d. x 100 mm)
SOURCE 15PHE, 96 ml
From the previous step, adjusted to
1.0 M ammonium sulphate, and 0.5 litres/cycle
was applied
1.0 M ammonium sulphate,
50 mM phosphate, pH 7.4
50 mM phosphate, pH 7.4
0 to 45% B, 15 column volumes
200 cm/h
Buffer A:
Buffer B:
Gradient:
Flow:
A 280nm
Åke Danielsson, Ingemar Daniels, Makonnen Belew, Bo Forsberg and Hans J. Johansson,
Amersham Pharmacia Biotech AB, SE-751 84 Uppsala, Sweden
Purpose
To achieve consistent scale-up at the polishing stage of a downstream process, with different
chromatography techniques.
Introduction
The main aim at the final, polishing stage of a downstream process for a biopharmaceutical is to remove
trace contaminants such as structural variants of the product. To achieve consistent scale-up from the lab
bench to the production hall of these difficult separations can be a major bottleneck in process development.
A 280nm
0.50
0.15
0.40
0.30
We have used SOURCE™ chromatography media based on monosized 15 and 30 µm divinylbenzene/polystyrene beads for this task (Fig. 1). Using these media, the required high resolution was achieved at low or
moderate operating back-pressures (Fig. 2-3).
0.10
0.20
Scaled-up separations with SOURCE media for RPC, HIC and IEX are shown in Figures 4-9. Scale-up was
facilitated by the use of a uniform control platform, UNICORN™, which has a common user interface
for all scales of operation.
0.05
0.10
Pool
Pool
0.00
0.0
0.00
2.0
4.0
6.0
8.0
10.0
12.0
Volume (litres)
Fig 7. Purification of r-exotoxin A on SOURCE 30Q.
Recovery of exotoxin A was 92% (by immunodiffusion).
0
20
40
60
Time (min)
Fig 8. Polishing of r-exotoxin A on SOURCE 15PHE.
A 280nm
Low back-pressure
A 280nm
0.40
a
b
0.30
Back-pressure in bar
Back-pressure in bar
25
40
Bed height
0.30
20
0.20
0.20
15 cm
10 cm
5 cm
3 cm
30
25
15
0.10
0.10
35
SOURCE 30
Other 1
Other 2
20
10
0.00
15
0.00
0.0
2.0
4.0
6.0
8.0
Volume (ml)
0.0
Fig 9. HPLC analysis of r-exotoxin A (a) before and (b) after polishing on SOURCE 15PHE.
2.0
4.0
6.0
10
8.0
Volume (ml)
5
5
0
0
0
Summary
Polishing separations on SOURCE 15 and SOURCE 30 media were consistently scaled-up from
laboratory scale to pilot, and production scale.
Monosized SOURCE 30 media gave back-pressures below 10 bar at flow rates up to 1800 cm/h
(10 cm bed height, water, room temperature).
Consistent resolution was obtained at 300 cm/h and 1000 cm/h when 100 mm i.d. and 800 mm
i.d. FineLINE production columns packed with SOURCE 30S were compared.
RAK design AB.
SOURCE,UNICORN, Sepharose, RESOURCE, FineLINE, BioPilot, BioProcess and STREAMLINE are trademarks of Amersham
Biosciences Limited or its subsidiaries. Amersham Biosciences is a trademark of Amersham plc.
Amersham Biosciences Björkgatan 30, SE-751 84 Uppsala,
Sweden. Amersham Biosciences UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England.
Amersham Biosciences 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855 USA. Amersham Biosciences
Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany. Amersham Biosciences K.K. Sanken Building,
3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan. All goods and services are sold subject to the terms and
conditions of sale of the company within the AmershamBiosciences group that supplies them. A copy of these terms
and conditions is available on request. © Amersham Biosciences 2000 – All rights reserved.
Fig 1. Electron micrograph of SOURCE 30
beads. Note the uniform size and absence
of fines, fragments, and broken beads.
18-1124-77
Edition AB
400
800
1200
1600
2000
Linear flow in cm/h
Fig 2. Pressure/flow rate characteristics of
SOURCE 30. The monosized SOURCE 30
matrix was compared with polysized matrices
on the market. (Other 1 is a nominally 50 µm
diameter particle; Other 2 is a nominally
35 µm particle. Both were handled according
to the manufacturers recommendation.)
0
200
400
600
800
1000
Linear flow in cm/h
Fig 3. Back-pressure of SOURCE 15 in
large-scale columns. The experiments
were performed in a FineLINE™ 100
column (100 mm i.d.).