The use of NaOH for CIP of rProtein A media: a 300 cycle study
Hans J Johansson1, Annika Bergenstråhle2, Gustav Rodrigo2 and Katarina Öberg2
1
Amersham Biosciences, Piscataway, NJ, USA. 2Amersham Biosciences, Uppsala, Sweden
Introduction
Experimental, 300 cycles
Due to its unique selectivity, high recovery and
productivity, Protein A affinity chromatography is
the preferred technique for commercial purification
of therapeutic antibodies. The functional lifetime of
a Protein A resin is one of the factors having a
substantial impact on the overall economy of an
affinity step. It is thus of great importance to develop
efficient purification protocols that meet set quality
criteria while allowing reuse for a large number of
cycles. The objective of this study was to develop
an effective protocol for CIP (cleaning in place) of
MabSelect™, an agarose based Protein A media,
developed for large-scale capture and purification of
monoclonal antibodies from cell culture supernatants.
The feedstock used was the supernatant of a CHO (Chinese hamster ovary) cell
line expressing a humanized IgG. The study was designed using a strategy where
5 complete purification cycles were followed by a CIP cycle. The dynamic capacity
at 1% break through was determined to be 18 mg/ml medium at a linear velocity
of 300 cm/h.
Cycle 1
Cycle 299
The choice of appropriate elution and regeneration
procedures followed by sodium hydroxide/sodium
chloride combinations for CIP made it possible to
design a protocol that delivered a consistent high purity
and recovery using a MabSelect resin for 300 cycles.
With proper adjustments of the specific target antibody elution conditions the protocol in this paper
should be a good starting point for a majority of
antibodies expressed by mammalian cell cultures.
The relatively high resistance of the Protein A resin
to the tested CIP solutions suggests that an alternative
protocol could include the use of NaOH/NaCl in
every cycle.
18-1177-64 AA
Cycle179
B001:UV1_280nm
B250:UV1_280nm
Cycle 239
B100:UV1_280nm
B300:UV1_280nm
B150:UV1_280nm
BCellSup001:UV1_280nm
B1001:Inject
B1001:UV1_280nm
B250:UV1_280nm
B200:UV1_280nm
B100:UV1_280nm
B300:UV1_280nm
B150:UV1_280nm
BCellSup001:UV1_280nm
B1001:Inject
B200:UV1_280nm
mAU
60.0
1000
6000
50.0
800
5000
40.0
4000
30.0
600
3000
20.0
10.0
2000
Purification cycle:
200
0.0
1000
0.0
10.0
20.0
30.0
40.0
50.0
60.0
Selection of chromatograms from 300 cycle study: 1, 59, 119, 179, 139 and 299.
cycle 5
pH
Cycle 150
0.0
ml
5.0
10.0
15.0
20.0
25.0
ml
Analytical gel filtration (chromatograms super imposed) of product from cycle 1, 100,
150, 200, 250, 300 and cell culture supernatant. Experimental: Superdex™ 200
HR10/30, 100 µl load, 0.6 ml/min, UV 280 nm.
Cycle 300
0.0
1800
1400
3000
Cycle description for studies with solutions 1-3, 6:
Linear flow velocity: 336 cm/h
Binding buffer:
3 CV
Elution buffer:
4 CV
CIP, see above:
8.8 CV
Binding buffer:
4 CV
Initially, and after every 10 cycles dynamic binding capacity was measured.
ml
The recoveries were between 95–100%
throughout the study. The IgG dimer content
was between 0.5–1.5%, the variation probably
being a function of different storage times in
the low pH elution buffer.
1200
1000
800
400
1000
200
0
1
0
20
0
Waste
40
60
80
0
ml
Selection of chromatograms with subsequent CIP included, Cycle 5, 150 and 300.
50
100
150
200
250
300
Run number
Analysis of the CHOP content (Chinese hamster ovary proteins) in the purified product.
Description for studies with solutions 4 and 5:
Results
Linear flow velocity: 336 cm/h
Binding buffer:
3 CV
Elution buffer:
4 CV
Binding buffer:
4 CV
CIP, see above:
8.8 CV
Binding buffer:
4 CV
Initially, and after every 20 cycles dynamic binding capacity was tested.
35
flow 1.1 ml/min
flow 1.1 ml/min
flow 1.1 ml/min
flow 1.1 ml/min
flow 1.1 ml/min
flow 1.1 ml/min
25.0
Results
1600
4000
Investigated solution:
8.8 CV- contact time 16 min
8.8 CV- contact time 16 min
8.8 CV- contact time 16 min
8.8 CV- contact time 16 min
8.8 CV- contact time 16 min
8.8 CV- contact time 16 min
20.0
2000
The studies were done in HR 5/10 Columns** (5 x 100 mm) packed with MabSelect.
Determination of dynamic binding capacity of hIgG at 10% breakthrough has been
used for analysis.
10 mM NaOH, 1 M NaCl
50 mM NaOH, 1 M NaCl
100 mM NaOH, 1 M NaCl
250 mM NaOH, 1 M NaCl
50 mM NaOH, 1 M NaCl
50 mM NaOH
15.0
5000
Experimental, NaOH/NaCl stability
25 mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.1
100 mM acetic acid, pH 2.8
hIgG ( Gammanorm, Pharmacia & Upjohn) 2.5 mg/ml
10.0
Analyical gel fitration enlarged.
600
All breakthrough experiments were performed using:
5.0
mAU
2000
– 5.5 Cv CIP buffer 50 mM NaOH, 1 M NaCl,
– 6 Cv regeneration buffer
– 4 Cv equilibration buffer
The MabSelect resin was packed in a HR 5/10 column** (bed dimensions 5x95 mm)
Binding buffer:
Elution buffer:
Sample:
-10.0
0
0
CHOP (ng/mg)
– 2 Cv equilibration and wash buffer: 25 mM Tris, 25 mM NaCl, 5 mM EDTA,
pH 7.1
– 12 Cv CHO cell culture supernatant, corresponding to a load of
17 mg Mab/ml MabSelect
– 7 Cv equilibration and wash buffer
– 6 Cv elution buffer: 0.1 M acetic acid, the collected fraction was adjusted to
pH 5–6 with 1.5 M Tris before freezing and storage for analysis
– 3 Cv regeneration buffer: 0.4 M acetic acid, 0.5 M NaCl, 0.1 % Tween 20
– 3 Cv equilibration buffer
10 mM NaOH + 1 M NaCl (solution 1): No significant decrease
in capacity after 300 cycles.
50 mM NaOH + 1 M NaCl (solution 2 and 5): The decrease in
DBC after 100 cycles was 0%, after 200 cycles 4%, and after
300 cycles 11%.
In contrast, when 50 mM NaOH without NaCl was tested as
CIP solution the decrease in DBC after 61 cycles was 16%.
These indicate that the addition of NaCl to the NaOH solution
has a significantly positive effect on the NaOH stability of
rProtein A.
100 mM NaOH + 1 M NaCl (solution 3) and 250 mM NaOH
+ 1 M NaCl (solution 4): The decrease in DBC for solution 3 was
16% after 163 cycles and for CIP solution 4, 18% after 100 cycles.
In many processes a lifetime of 100 to 200 cycles will be more
than economically sufficient. In those cases, solutions (3 & 4),
or even higher concentrations might very well be considered.
35
30
30
25
Dynamic bindning capacity (10%)
Conclusion and Discussion
Cycle119
mAU
Dynamic bindning capacity (10%) mg/ml
• Dynamic binding capacity at 10% breakthrough of > 30 mg
human IgG at a flow velocity of 500 cm/h in a column packed
to 20 cm bed height.
• Recombinant "green"* Protein A coupled to the matrix via a
C-terminal cysteine. Mean particle size of 85 µm.
• Highly cross-linked agarose beads allowing linear flow rates
up to 700 cm/h.
* No animal derived material used in the fermentation and
purification of the recombinant Protein A used for production
of MabSelect.
Cycle 59
400
CIP cycle:
Media Characteristics
Conductivity
mAU
20
15
50 mM NaOH + 1 M NaCl
50 mM NaOH + 1 M NaCl
50 mM NaOH without NaCl
10
25
50 mM NaOH + 1 M NaCl
50 mM NaOH + 1 M NaCl
100 mM NaOH + 1 M NaCl
10 mM NaOH + 1 M NaCl
250 mM NaOH + 1 M NaCl
50 mM NaCl without NaCl
20
15
5
10
0
0
10
20
30
40
50
60
70
80
Number of cycles
90
100
0
50
100
150
200
250
300
350
Number of cycles
Comparison between 50 mM NaOH with and without 1 M NaCl.
** Columns were equipped with 20–80 µm Vyon™ filters.
Vyon is a trademark of Porvair plc
MabSelect and Superdex are trademarks of Amersham Biosciences plc