Protein A Resin:
Sanitization and CIP
Sheldon E. Broedel, Jr., Ph.D.
Athena Enzyme Systems Group
Cleanliness is Next to …
• Purpose is to purify an IgG molecule
– Separate from upstream matrix
• Do not contaminate
– Resin should not introduce contaminants
• Used as the capture step
– Complex feed stream
• Two schools of thought
– Single use
– Multiple use …. BUT!
Column Cleaning
• Purpose
– Remove unwanted agents while not
destroying the resin
• Sanitization
– Removal of viral, bacterial, fungal and
parasitic organisms
• CIP – Cleaning In Place
– Removal of any adventitious agents (proteins,
lipids, nucleic acids, etc.)
Operational Compatibility
• Ligand – Protein A bound to solid support
– Retain ligand binding activity
– Minimize leakage
• Backbone – polymeric material on which
the resin is built
– Chemically compatible
• Disposal, cost and regulatory
considerations
Most Commonly Used: NaOH
• Used at 0.1 to 0.5 M
– Alkaline pH, highly chaotropic, chemically
reactive
• Broad spectrum cleaning agent
–
–
–
–
–
Proteins: dissolves, denatures, disrupts
Nucleic Acids: dislodges from resins
Lipids: saponifies rendering them soluble
Anti-microbial: Kills viruses, bacteria, yeast, fungi
Inactivates prions and endotoxin
NaOH Sanitization
Organism
NaOH (M)
Time to >3
log Kill
Temp (ºC)
Viruses
0.1
<1 h
22
E. coli
0.01
2
4 or 22
S. aureus
0.1
1
4 or 22
C. albicans
0.5
1
4 or 22
A. niger
0.5
1
4 or 22
B. subtilis spores
1.0
48
22
P. aeruginosa
0.5
1
22
Adapted from Application Note 18-1124-57 AG, GE Healthcare, 2009
Inactivation of Endotoxin
Endotoxin (ng/ml)
120
100
0.1 M NaOH
80
0.5 M NaOH
60
1.0 M NaOH
40
20
0
0
10
20
30
40
Time (hours)
Adapted from Application Note 18-1124-57 AG, GE Healthcare, 2009
50
60
Challenges with Protein A Resins
• Protein A is a protein
– susceptible to denaturing by OH
• Protein coupled to backbone
– linkage susceptible to cleavage
• Incompatible backbones (CPG resins)
• Need an alternative approach
Process Cycle
Equilibration
Neutral Buffered Saline
Load
Serum, Culture Supernatant, Cell-free Extract
Wash
Neutral Buffered Saline
Elute
Low pH Buffer
Regeneration
CIP Buffer
Concepts to Consider
• Resin Lifetime
– Critical to ensure robustness of process
– Resin yields reliable quality
• Chemical compatibility with solid support
• Performs desired cleaning and sanitization
• Use scale-down model
Attributes to Measure
• Lifetime test model
– Repeated cycles of use with and without a
load
– Employ different CIP solutions and exposure
times
• Measure
– Product yield
– Change in dynamic capacity
– Adventitious agent removal
Example: Loss of Dynamic Capacity
Top Graph:
rProtein A Sepharose FF
Bottom Graph:
MabSelect
CIP1: 3CV 0.1 M NaOH
CIP2: 2CV 0.1 M phosphoric
acid, 1CV water and 3 CV
50 mM NaOH, 0.5 M NaCl
CIP3: 3CV 50 mM NaOH, 0.5 M
NaCl
Taken from Jiang et al. 2009.
Example: Leakage and HCP
MabSelect Xtra
[CIP: 5CV 0.5 M acetic acid, 0.1
M Na2SO4; 5CV PBS; 5CV 50
mM NaOH, 0.5 M NaCl]
Cycle
MabSelect SuRe
[CIP: 3.3CV 0.5 M NaOH]
Leakage
(ng/ml)
HCP
(ng/ml)
IgG Elute
(mg/ml)
Leakage
(ng/ml)
HCP
(ng/ml)
IgG
(mg/ml)
2
30.16
1,968
5.1
10.64
3,025
3.8
10
92.02
1,792
5.7
5.21
1,872
4.1
24
195.4
1,288
10.34
1,736
25
0.58
0
NA
0.33
0
NA
40
111.1
580
5.7
10.44
1,924
4.1
49
42.79
616
5.5
9.81
1,768
4.4
50
0.3
0
NA
0.34
0
NA
Adopted from Hahn et al. 2006.
Example: Alternative to NaOH
• ProSep-vA HC – a CPG resin
• Previous work had shown that 0.3% HCl pH 1.5
followed by 6 M Guanidine HCl every 5th cycle
was inadequate
– Leakage and HCP significantly higher that agarosebased resins
• Devised a new CIP solution by modeling
– Panel of microbes
– Performed challenge experiments
• CIP:
– 4CV 132 mM phosphoric acid, 184 mM acetic acid,
2.2% benzyl alcohol hold for 3 hour every 5th cycle.
Example: Alternative to NaOH
Cycle Number
Yield (%)
HCP (ng/mg)
CHO DNA
(ng/mg)
Protein A
(ng/mg)
30
97
1,390
19
14
60
98
1,400
15
18
90
98
1,580
6
18
110
95
1,400
5
23
130
97
1,350
4
27
160
101
1,950
9
16
210
96
1,720
NT
13
260
95
1,750
NT
17
300
93
1,680
19
29
From Rogers et al. 2009.
Summary
• CIP protocol depends on resin selected for process
– 0.5 M NaOH -for hydroxide-resistant resins
– 50 mM NaOH, 0.5 M NaCl with acid stripping for
non-resistant resins
– Phosphoric-acetic acid, benzyl alcohol mix for nonagarose resins
• Should be validated for suitability
– Operational Parameters: exposure duration, flow rate,
temperature, etc.
– Performance Characteristics: HCP clearance, Protein
A leakage, yield, dynamic capacity, etc.
References
• Janson, J.-C. 2011. Protein Purification: Principles, High
Resolution Methods and Applications, 3rd Ed, Vol. 54. John
Wiley & Sons, Hoboken, NJ. ISBN 978-0-471-74661-4.
• Hober et al. 2007. J. Chromato. A. 848:40-47.
• Rogers et al. 2009. J. Chromato. A. 1216:4589-4596.
• Jiang et al. 2009 J. Chromato. A. 1216:5849-5855.
• Hahn et al. 2006. J. Chromato. A. 1102:224-231.
• Swinnen et al. 2007. J. Chromato. B. 848:97-107.
• McCue et al. 2003. J. Chromato. A. 989:139-153.
• Application Note 18-1124-57 AG, GE Healthcare, 2009.
• Johansson et al. 2009. Application Note 18-1177-64 AA, GE
Healthcare.
• Antoniou and Carter. 2006. BioPharm Intl.