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

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Endo

toxi

n (n

g/m

l)

Time (hours)

0.1 M NaOH 0.5 M NaOH 1.0 M NaOH

Adapted from Application Note 18-1124-57 AG, GE Healthcare, 2009

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

Elute Low pH Buffer

Wash Neutral Buffered Saline

Load Serum, Culture Supernatant, Cell-free Extract

Equilibration Neutral Buffered Saline

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

Taken from Jiang et al. 2009.

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

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]

MabSelect SuRe [CIP: 3.3CV 0.5 M NaOH]

Cycle 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 agarose-

based 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 non-

agarose 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.