1. sports
2. skating
3. figure skating

Design of a novel agarose-based resin platform
Hans J Johansson,* Hans Berg, Patrick Gilbert, Mark Hicks, Caroline Tinsley, Duncan Sinclair. Purolite Research and Development. Llantrisant, Wales.
*[email protected]
Introduction
Results
Selectivity
Monoclonal antibody purification*
• The first agarose based chromatography beads were introduced by
Hjerten in 1962. Fifty years later beaded agarose has become the
dominant resin for protein purification and is extensively used, from
research scale in sub mL volumes, to full scale manufacturing in
500 litre chromatography columns.
Dynamic binding capacity (DBC), pressure/flow properties, and selectivity
are three key performance parameters for any process resin.
The charged groups of the SP and Q ligands used in Praesto media are
identical to the charged groups used in many other ion exchange resins.
Minor differences in selectivity still occur due to differences in base
matrix, ligand density and the presence or absence of surface extenders.
The Praesto platform demonstrates selectivity very similar to established
agarose-based resins with the SP (Fig. 4a and 4b), the exception being
Capto S, a dextran grafted resin, that shows poorer resolution for this
application.
Cation exchangers are present in most antibody processes with the
primary function to remove product variants such as high molecular
weight (HMW) aggregates and fragments. Here we compare four different
cation exchangers with respect to DBC and removal of high molecular
weight aggregates and host cell proteins. The generic conditions used
were the same for all four resins and not individually optimized for each
cation exchange resin.
800
800
600
400
0
Figure 1– Selectivity (Kav) curves
0.8
0.7
0.7
0.7
0.6
0.6
0.6
0.5
0.5
0.3
Praesto Pure45
0.3
Praesto Pure65
0.3
Praesto Pure90
0.2
Sepharose 4 FF
0.2
Sepharose 4 FF
0.2
Sepharose 4 FF
0.1
Sepharose 6 FF
0.1
Sepharose 6 FF
0.1
Sepharose 6 FF
4.5
5
logMW
5.5
6
0
4
4.5
5
logMW
3.5
3
4
4.5
5.5
6
0
4
4.5
5
0.5
2
1.5
1
2.5
Praesto SP90 (90 μm)
Praesto SP65 (65 μm)
Capto S (90 μm)
Capto SP ImpRes (40 μm)
SP Sepharose Fast Flow (90 μm)
Praesto SP45 (45 μm)
Praesto SP65 (65 μm)
SP Sepharose High Performance (34 μm)
3.5
3
4
4.5
Praesto SP65, Praesto SP45, SP Sepharose High
Performance and Capto SP ImpRes were packed
at 4 bar to a bed height of 20 cm in a HiScale 26/40
column.
50
40
1000
30
0
20
10
15
Retention volume (mL)
5
25
30
60
1500
1000
80
90
70
60
50
40
6
8
Capto SP ImpRes
Praesto SP90
Praesto SP45
Capto S
Praesto SP65
Conductivity
Conductivity
25
30
50
RT 2.4 min
RT 6 min
2
4
Residence time (min)
Q Sepharose FF
Capto Q ImpRes
6
Figure 3b – Intermediate purification and polishing:
comparison of DBC at different residence times for
Praesto Q65, Praesto Q45 and Capto Q ImpRes.
Column: Tricorn™ 5/50
Run conditions same as in 3a.
130
0
65
6
6.00
5
5.00
4
3
2
0
0.00
0.00
0.00
20.00
60.00
80.00
100.00
71
Capto S ImpAct
Capto S ImpAct
Praesto SP45
Praesto SP45
Praesto SP65
Praesto SP65
Capto ImpRes
Capto ImpRes
10
Praesto A prototype 2 (large pore)
3 minutes RT (DBC 10%)
38
52
50
Resin
Figure 5 – DBC measured with a 5 mg hIgG/mL,
pH 7.4, solution.
75
100
90
80
70
50
8
6
4
Praesto PtA
MabSelect SuRe
60
2
75% cut off
0
5
10
15
20
25
30
35
120.00
Recovery of protein %
Figure 9 – Host Cell Protein (HCP) clearance for MAb A
Relative alkaline stability comparison between MabSelect SuRe
and Praesto Protein A prototype.
55
10 minutes RT (DBC 10%)
40.00
12
48
25
120.00
Figure 8 – Protein recovery vs. cumulative HMW aggregate removal at 10 and 70 % of the determined DBC.
Elution with linear gradient over 20 column volumes from 20 mM sodium acetate, pH 5.0 to 20 mM acetate
+ 0.5 M sodium chloride, pH 5.0. The starting material (Protein A purified MAB) had a HMW aggregate
content of 4 %.
Figure 6 – Alkaline stability
Praesto A prototype 1 (small pore)
3 minutes RT (DBC 10%)
33
0
100.00
40
Number of CIP cycles
Figure 6 – CIP study over 40 cycles using 1.0 M NaOH,
30 minutes/cycle. DBC measured with a 5 hIgG/mL
solution, pH 7.4.
0
Capto S ImpAct
Purolite Praesto SP45
MAb A RE1 (10% DBC)
Load
39.0
39.0
F1
0.44
0.25
F2
0.51
0.74
F3
0.70
0.75
F4
0.70
0.91
F5
0.96
0.66
F6
0.76
1.12
F7
0.99
1.07
F8
1.30
Below LOD
Table 1 – Conditions used: Approximately 500 mg MAb A/mL resin was loaded under non-binding
conditions (Protein A eluate pH adjusted to 7.7) at two different residence times. 2.4 and 6.0 minutes.
The column bed height was 6 cm.
• Purolite successfully developed homogeneous agarose resins with
excellent pressure/flow properties, capacity and resolution
Praesto is a trademark of Purolite Corporation
ÄKTA, Tricorn, Capto, MabSelect SuRe, and Sepharose are trademarks of GE Healthcare companies
Recovery of protein %
39
6 minutes RT (DBC 10%)
80.00
(ng/mL)/mg/mL
*The monoclonal antibody purification evaluation was performed by
Professor Anurag Rathore at the Indian Institute of Technology, Delhi.
2.00
Flow rate: 0.3 mL/min
60.00
(ng/mL)/mg/mL
• Alkaline stable Protein A prototype resins with a capacity matching the
market leading Protein A resin, MabSelect SuRe, has been designed
3.00
1.00
40.00
Rt 6.0 minutes
• A dynamic binding capacity of > 120 mg/mL was achieved for two
different antibodies using Praesto SP45
ImpAct
4.00
1
20.00
Rt 2.4 minutes
Summary and conclusion
MAb A – Protein recovery vs. cumulative aggregate % at 70% DBC.
Elution buffer: 50 mM sodium acetate,
1 M NaCl, pH 5.
6 minutes RT (DBC 10%)
130
DBC MAb B at 5% breakthrough (mg/mL of resin)
MAb A – Protein recovery vs. cumulative aggregate % at 10% DBC.
Run conditions same as in 4a.
6 minutes RT (DBC 10%)
50
Figure 8 – HMW aggregate removal for MAb A
Column: Tricorn 5/50
8
Figure 3a – Capture and intermediate purification:
comparison of DBC at different residence times for
Praesto Q90, Praesto Q65 and Q Sepharose 6FF.
RT 6 min
123
65
30
Figure 7 – DBC data for two different monoclonal antibodies of subclass IgG1. MAb A: Protein A eluate
adjusted to pH 5.5 with 1.75 M acetic acid. MAb B: 20 mM acetate, pH 5.0.
110
0
RT 2.4 min
79
0
Figure 4b – Intermediate purification and polishing:
comparison of selectivity of Capto SP ImpRes,
Praesto SP45, and Praesto SP65.
MabSelect SuRe
3 minutes RT (DBC 10%)
81
Capto S ImpAct
DBC MAb A at 5% breakthrough (mg/mL of resin)
Figure 4a – Capture and intermediate purification:
comparison of selectivity of SP Sepharose Fast
Flow, Praesto SP90, and Capto S.
Comparison between MabSelect SuRe™ and two different
Praesto Protein A prototypes.
Praesto Q65
Flow rates: 0.42 mL/min (2.4 minutes residence
time), 0.17 mL/min (6 minutes residence time)
20
10
15
Retention volume (mL)
50
RT 6 min
92
Capto S ImpAct
A new generation Protein A resins
60
Praesto Q90
Sample buffer: 50 mM Tris (pH 8.5)
5
Sepharose 6 Fast Flow
Figure 5 – Dynamic binding
capacity
Praesto Q45
Sample load: Until 10% breakthrough
0
RT 2.4 min
80
RT 6 min
System: ÄKTA™ Pure 25L
70
30
RT 2.4 min
Residence time: 4 minutes
80
129
Capto SP ImpRes
Capto SP ImpRes
10
0
86
RT 6 min
123
0
Purolite has started the design of an alkaline stable Protein A resin.
Preliminary results indicate that dynamic binding capacities well above
50 g/L with a new alkaline stable Protein A can be achieved.
Praesto Q65
Sample: 10 mg/mL Bovine Serum Albumin
20
500
40
30
4
Residence time (min)
30
Start buffer: 50 mM sodium acetate, pH 5.
Comparison of DBC for Praesto Q45, Praesto Q65 and
Capto Q ImpRes anion exchangers.
100
2
50
40
0
RT 2.4 min
101
RT 6 min
Sample volume: 0.5 mL (5 x 50 mm)
Figure 3b – Intermediate
purification and polishing
90
0
70
2000
20
500
85
Praesto SP45
RT 2.4 min
Sample: 25 mg/mL IgG, 5 mg/mL Lactoferrin in
50 mM sodium acetate buffer solution, pH 5.
With the continuing development of high titer cell lines, high capacity is
becoming increasingly important. The Praesto range of ion exchangers
have significantly higher dynamic binding capacity compare to other
non-grafted ion exchange resins.
20
1500
RT 6 min
89
Praesto SP45
Column volume: 1 mL
Dynamic binding capacity
logMW
60
0
Praesto SP90, Praesto SP65, and Capto S were
packed at 4 bar to a bed height of 20 cm in a
HiScale™ 26/40 column.
6
70
2000
44
80
10
Figure 2b – The figure compares the pressure/flow
properties of Praesto SP65, Praesto SP45, SP
Sepharose High Performance and Capto SP ImpRes.
Comparison of DBC for Praesto Q65, Praesto Q90 and
Q Sepharose Fast Flow anion exchangers.
5.5
0
Pressure (bar)
Figure 3a – Capture and
intermediate purification
0.4
0.4
4
2.5
DBC 10% breakthrough (mg/mL)
0.8
0
2
1.5
0
2500
RT 2.4 min
63
RT 6 min
90
80
400
Figure 2a – The figure compares the pressure/flow
properties of Praesto SP90, Praesto SP65, SP
Sepharose Fast Flow and Capto S.
DBC 10% breakthrough (mg/mL)
0.8
Kav
0.9
0.9
Kav
Kav
Selectivity curve of Praesto Pure90,
compared with Sepharose 4 Fast Flow
and Sepharose 6 Fast Flow, obtained with
RNase, Bovine Serum Albumin, Ferritin
and Thyroglobulin.
0.9
0.4
1
SP Sepharose Fast Flow were packed at 2 bar to
a bed height of 20 cm in a HiScale 26/40 column.
1
2500
Praesto SP65
RT 2.4 min
100
90
Pressure (bar)
To cover applications from capture to polishing three different particle
sizes all with a porosity optimal for medium sized proteins (50 - 200 kD)
were designed.
0.5
0.5
3000
100
Fractions
5% DBC data for MAb B on four resins at two different residence times.
Praesto SP65
ng HCP/mg MAb
0
Minimal matrix volume (5 - 7 %): possibility to design high capacity resins
without the use of grafting technology.
1
3000
200
Alkaline stable: allows the use of high concentrations of sodium
hydroxide for cleaning and sanitization in place.
Selectivity curve of Praesto Pure65,
compared with Sepharose 4 Fast Flow
and Sepharose 6 Fast Flow, obtained with
RNase, Bovine Serum Albumin, Ferritin
and Thyroglobulin.
600
200
Extremely hydrophilic: minimal unspecific interaction, long functional
life time.
Protein separation of 25 mg/mL IgG and 5 mg/mL Lactoferrin over
Praesto SP45, Praesto SP65 and Capto SP ImpRes.
UV signal (mAU)
Agarose was chosen as the base for the Praesto™ platform because of
the inherent properties of agarose:
1000
Linear velocity (cm/h)
Linear velocity (cm/h)
1200
Protein separation of 25 mg/mL IgG and 5 mg/mL Lactoferrin over
Praesto SP90, SP Sepharose 6 Fast Flow and Capto S.
5% DBC data for MAb A on four resins at two different residence times.
Strong anion exchangers are commonly used as a scavenger step in flow
through mode to remove trace contaminants and ensure sufficient virus
clearance. In this study we looked at the removal of host cell proteins
under non-binding conditions on Praesto Q65. All fraction showed HCP
levels of 1 ppm or lower.
Table 1 – Praesto Q65
Figure 7 – Dynamic binding capacity at 5 % breakthrough
Cumulative HMW % of total
1400
Designing a platform
1
Pressure/flow performance of Praesto SP65, Praesto SP45,
SP Sepharose High Performance and Capto SP ImpRes.
Figure 4b – Cation selectivity,
intermediate purification and
polishing
UV signal (mAU)
Pressure/flow performance of Praesto SP90, Praesto SP65,
SP SepharoseTM Fast Flow and CaptoTM S.
Figure 4a – Cation selectivity,
capture and intermediate
purification
DBC at 5% breakthrough (%)
• The result is a novel range of homogeneous agarose resins designed
for large scale manufacturing. The resins show significantly improved
pressure/flow properties, capacity, and resolution compared to what
is available today.
Selectivity curve of Praesto Pure45,
compared with Sepharose™ 4 Fast Flow
and Sepharose 6 Fast Flow, obtained with
RNase, Bovine Serum Albumin, Ferritin and
Thyroglobulin.
Figure 2b – Intermediate
purification and polishing
Figure 2a – Capture and
intermediate purification
Conductivity (mS/cm)
• In this paper we have used a new technology to produce beaded
agarose of different particle sizes ranging from 40 - 90 μm. The
objective has been to improve pressure flow properties while
creating a porosity structure optimal for protein chromatography.
Cumulative HMW % of total
Using a unique cross-linking chemistry the rigidity of Praesto resins allows
for high flow velocities at pressures relevant to large scale operation.
Conductivity (mS/cm)
Pressure/flow properties
Praesto Q65 for polishing of MAb A
at a load of 500 g/L resin
Purolite Praesto SP65
Capto S ImpRes
MAb A RE2 (70% DBC)
Figure 9 – There were no major difference in the HCP clearance ability under the conditions used. The
Protein A purified starting material had a HCP content of approximately 40 ng HCP/mg MAb.
The agarose development team