key interactions of polysorbates in protein formulations · • interfacial stresses may lead to...
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Key Interactions of Polysorbates in Protein Formulations
Hanns-Christian Mahler & Kishore RavuriPharmaceutical Development & Supplies, PTD Biologics EUHoffmann-La Roche AG, Basel, Switzerland
Surfactants are potent Stabilizers for Proteins against Interfacial Stresses
• “Interfacial stresses“ are encountered during many stages of production, shipment and use e.g.
– Air/liquid interfaces (e.g. shaking)
– Ice/Liquid interfaces (e.g. freezing and thawing)
– Surface interactions (e.g. manufacturing equipment)
• Interfacial stresses may lead to “protein instabilities“ such as adsorption, aggregation or precipitation (“particle formation“) Kiese et al, J. Pharm Sci. 2008, 2009
• Surfactants (such as the non-ionic Polysorbate 20 or 80) effectively protect Proteins against aggregation caused by interface-induced stresses and adsorption Carpenter, Arakawa et al. 1992; Kendrick et al. 1996; Kerwin, Heller et al. 1998; Randolph and Jones 2002; Mahler, Mueller et al. 2005; Kiese et al., 2008/2009
x+y+z+w=20O
O
O
OH
O
OOH
OOH x
y
z
10
O
w
Polysorbate 20
Fatty acid ester
6
6
O
OO
O
OH
O
OOH
OOH x
y
z
w
Polysorbate 80
1,4 anhydro-sorbitol
Poly-oxy-ethylene
Polysorbate 20 and 80 are the most widely used Surfactants in Protein Formulations
70% of marketed antibodies are formulated with Polysorbates Typical concentrations of PS20/PS80 range from 0.001-0.1% (w/v)
Effective protection of proteins against aggregation caused by interface-induced stresses and adsorption
Polysorbate concentration
Pro
tein
con
cent
ratio
n
How Polysorbates stabilize Proteins
Effect of mechanical stress on Mab T in the presence and absence of Polysorbate
0 % 0.005 % 0.01 % 0.02 % 0.05 % PS20
Polysorbate stabilize (many) Proteins against shaking-induced aggregation/precipitationKiese, S., Pappenberger, A. Friess, W., Mahler, H.-C. (2008) J. Pharm. Sci 97(10):4347-4366
• Note: the CMC of Polysorbates does not unequivocally correlate to the stabilization behaviourMahler, H.-C., Senner, F., Mäder, K., Müller, R. (2009) J.Pharm.Sci. 98(12):4525-33
Mechanism of Polysorbate Function(s)
Adsorption of polysorbate to interface: a surface tension treatmentKishore Ravuri, yet unpublished results
c[MAB]
c[MAB]
c[MAB]
Prot
ein
conc
entr
atio
n (m
g/m
L)
PolysorbatConcentration (μM)1 10 100 1000
[PS] < CMC[PS] < CMC[PS] << CMC [PS] > CMC[PS] = CMC[PS] = CMC
0
c[MAB]
c[MAB]
c[MAB] Antibody
Polysorbate
Interface
A
A
B
B
C
C
Langmuir adsorption isotherm adsorption equation
Stronger adsorption of polysorbate 20 observed in surface tension isotherm demonstrating that the stabilizing ability of the surfactant is surface energy driven.
Interaction of non-ionic surfactants with proteins: Studies with ITCKishore Ravuri, yet unpublished results
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
-24
-22
-20
-18
-16
-14-0.8
-0.6
-0.4
-0.2
0.0
0 20 40 60 80 100 120 140 160
Time (min)
µca
l/sec
Inte
rgra
ted
hea
ts (
μcal
)
• Low binding constants (K= 102-103 M-1 )
• Comparable to literature e.g. Garidel et.al.
• The above results confirms that interactions between surfactant and protein are non specific.
• Stabilizing effect of non-ionic surfactants is NOT interaction driven.
ITC titration of a non-ionic surfactant into a 10mg/mL mAb formulation, 20mM His/HCl, pH6, 25 °C
Synthesis of Polysorbates and Polysorbate Heterogeneity
NaOH,NaLaurate/NaOleate
H2O2
Phosphorous oxyacids
H. Vu Dang et al. / J. Pharm. Biomed. Anal. 40 (2006) 1155–1165
OHOH
OH
OH
OH
OH
O
OH OH
OH
OH
OO
O
OH
OH
OOH
OOH x
y
z
w
OO
O
OH
OCOR
OOH
OOH x
y
z
w
O
Fatty acids
1,4 sorbitan
Industrial Synthesis of Polysorbates
OO
O
OH
OCOR
OOH
OOH x
y
z
w
Desired molecule!
LC-MS TIC of polysorbate
OO
O
OCOR
OCOR
OOH
OOH x
y
z
w
On H
O n COR O
O
O
O
O
OH
OH n
n
O
O
O
OO
O n
n
COR
COR
PEG, Fatty acid/salts
OO
O
OH
OH
OOH
OOH x
y
z
w
O
O
O
OO
O n
n
OH
OH
On H
O n OH O
O
O
O
O
OH
OH n
n
• High batch to batch & supplier variabilityH. Vu Dang et al. J. Pharm. Biomed. Anal. 40 (2006) 1155–1165
Side Products in Synthesis of Polysorbates
Some challenges in the context of the use of Polysorbates in Protein Formulations
1. Adsorption of Polysorbate
Polysorbates can significantly adsorb to some filtersMahler, H.-C., Huber, F., Ravuri, S.K.K., Reindl, J., Rückert, P., Müller, R. (2010) J. Pharm. Sci.
• Dilution effects in filters (e.g. due to residual water), observed via parallel change of protein concentration and conductivity
• Adsorption of Polysorbate 80 to filters (>80% recovery after 3L pre-rinse with Product)
Filtrate volume (L)
0
2
4
6
8
10
12
0 1 2 3 4 5
0
1
2
3
4
5
Protein conc.
Pro
tein
co
nc. (
mg/
mL
)
Filtrate volume (L)
Conductivity Co
nduc
tivity
(m
S/c
m)
0
2
4
6
8
10
12
0 1 2 3 4 5
0
20
40
60
80
100
rel. Polysorbate 80
rel.
Po
lyso
rba
te 8
0 (
%)
Conductivity
Co
nd
uct
ivity
(m
S/c
m)
Some challenges in the context of the use of Polysorbates in Protein Formulations
2. Polysorbates in Formulations used for preclinical animal (Dog) studies
Pseudoallergenic reaction in dogs after administration of Polysorbate
• Polysorbate 20 (Tween 20) used for i.v. formulation of protein (stabilizer)
• A tox study was performed with rats and dogs: also placebo vehicle showed a pseudoallergic reaction in the dogs (well tolerated in rats and monkeys – as well as humans)
dogs to be excluded for tox studies with formulations containing polysorbates because hypersensitive compared to other species
• Potential mechanisms:
– Histamine-releasing properties of polysorbates Masini et al. (1985)
10 mg/kg of Polysorbate 80* caused severe hypotension after first administration and increase in plasma histamine
– Haemolytic properties of polysorbates Krantz et al. (1948!)
haemolysis of red blood cells in vitro due to the solubilizing activity of Tween 20 at concentrations > 100 mg/ 100 ml
Some challenges in the context of the use of Polysorbates in Protein Formulations
3. Degradation of Polysorbates in Bulk and/or aqueous formulation
Fatty acids
OO
O
OH
O
OOH
OOH x
y
z
10
O
w
Peroxides, formic and acetic acid
pH,
Temperature
Temperature
Light, O2, metals
Auto-oxidation Hydrolysis
1973
1978
1978
2007
2002
Polysorbates Can Undergo Degradation
Mechanistic picture Findings from investigations:Degradants isolated from placebo formulations stored at 25°C for 20 months
9
O
OO
O
OHO
OOH
OOH x
y
z
w
OHO
O
O
n
OH
OOH
OO
O
n
OHO
O
O
OHO
O
OHO
O
O
OHO
O
OHO
O
O
OH
O
OH
O
O
O
OH
O
OO
OH
O
O
OO
O
OO
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
O
HOH
O
H
O
aldehydes
ketones
alkanes
fatty acid esters
fatty acids
small molecules
Major degradants Minor degradants
Polysorbate 206
6
O
OO
O
OHO
OOH
OOH x
y
z
w
OHO
O O
O
OO
OO
OO
O
O
O
O
O
O
O
OH
O
HOH
OH
O
OH
O
O
O
O
O
O
O
O
O
Aldehydes Ketones
Alkanes
Fatty acidsSmall molecules
O
O
OH
O
O
OH
O
OH
O
Major degradants
Minor degradants
Polysorbate 80
Dominant Mechanisms In Polysorbate DegradationKishore, R.S.K., Pappenberger, A., Bauer Dauphin, I., Ross, A., Buergi, B., Staempfli, A., Mahler, H.-C. J. Pharm Sci., 2011, 100: 721
O
O O
O
OO
O
OOH
OOH
OOH
O
n
x
y z
m
H
H H
H
H
O
O O
O
OO
OH
OOH
OOH
OOH
n
x
y z
OH
Om
H+
-H+
+
0
0.2
0.4
0.6
0 4 8 12months
Poly
sorb
ate
(mg/
mL)
Profile of NMR signals of fatty acid in PS20
5 °C
25 °C
40 °C
Ester hydrolysis
Temp (°C) k (h-1) t1/2 (h)
PS20
5 9e-07 5.50e05
25 5e-05 1.39e04
40 2e-04 3.47e03
PS805 4e-07 1.73e06
25 5e-05 1.39e04
• t1/2 of Polysorbate20 hydrolysis at 40°C was about 5 months
• t1/2 of Polsyorbate20 hydrolysis at 5°C was negligible
Auto-oxidation of PEG
OOH
O O
C
O
OHHformic acid
OO
OOH
H
O
O
CH2 O
OH2
formaldehyde
O
OHH
formic acid
OO
OO
O
O
O
O OO
CH2 CH2
OO
Oacetaldehydeacetic acid
H
O
OH
O
Dominant Mechanisms In Polysorbate DegradationKishore R. S. K., Kiese S., Fischer S., Pappenberger A., Grauschopf U., Mahler H.-C. Pharm Res. 2011, 28:1194
Buildup of acids and aldehydes
Decker et al, Die Makromolekulare Chemie, 1974, 3531-3540
Storck et al, Die Makromolekulare Chemie, 1966, 50-73
9
O
OO
O
OHO
OOH
OOH x
y
z
w
OHO
OO
n
O
n
HH
R
OH C O OO
n
O
n
H
OHO
OO
n
O
n
OOH
O2
OO
n
O
n OH
HCHO+HCOOH
-1
fatty acids
• Auto-oxidation plays a dominant role in degradation of polysorbates. The rate of Hydrolysis is negligible at pharmaceutically relevant conditions (drug product storage of 5 °C & 25 °C )
• Along with rupture of PEG chains, there also occurs rampant degradation at the olefinic sites.
• It is likely that the radical initiation occurs first at the olefin site and then spreads to the PEG chains.
0 10 20
40
80
120
160
Con
cen
trat
ion
(μm
ol/L
)
Time (weeks)
Peroxide formation in placebos containing 0.02% PS20 and PS80 at 40 °C
Changes in PS20 and PS80 concentration measurement by micelle method over 1 year at 25 and 5 °C
Mechanistic PictureSummary
Impact of Polysorbate degradation on protein formulationsOverall considerations
• What is the impact of decreased surfactant content on the stability of the protein? Not enough stabilizer??
• What is the impact of decreased surfactant content on the stability of the protein? Not enough stabilizer??
Effect of decreased surfactant content
• What do the degradants do to the protein?• Do degradants impact product quality other
than interacting with protein?
• What do the degradants do to the protein?• Do degradants impact product quality other
than interacting with protein?
Effect of insoluble degradants from
hydrolysis
• What influence does auto-oxidation of PS have on the mAb?
• What influence does auto-oxidation of PS have on the mAb?
Effect of peroxides from auto-oxidation
Unshaken Shaken0.0
0.5
1.0
1.5
2.0
Unshaken Shaken0.0
0.5
1.0
1.5
2.0
Unshaken Shaken0
2
4
6
Mab340mo 5°C
Mab224mo 5°C
Solu
ble
Aggr
ega
te C
ont
ent
(S
E-H
PLC
(%
))
PS20 PS80
Mab112mo 5°C
Unshaken Shaken0
2
4
6
Unshaken Shaken0.0
0.5
1.0
1.5
2.0
Unshaken Shaken0.0
0.5
1.0
1.5
2.0
not tested
Initial Post Storage0.00
0.01
0.02
Initial Post Storage0.00
0.01
0.02
Initial Post Storage0.00
0.01
0.02
0.03
Mab340mo 5°C
Mab224mo 5°C
PS
Con
tent
(%
)
PS20 PS80
Mab112mo 5°C
Initial Post Storage0.00
0.01
0.02
0.03
Initial Post Storage0.00
0.01
0.02
Initial Post Storage0.00
0.01
0.02
not tested
Polysorbate content (by micelle assay)Soluble aggregates
Impact of Polysorbate degradation on protein formulationsEnd of shelf life shake stress
1E-4 1E-3 0.01
0
10
20
30
40
Surf
ace
Pres
sure
(mN
/m)
relative PS20 concentration (w/v)
1E-4 1E-3 0.01
0
10
20
30
40
relative PS80 concentration (w/v)
• Degradation products of PS still show surface activity even after 60% loss of content by micelle assay
PS20 at t0
Surface pressure measured in 20mM His/HCl with PS20 and PS80
PS20 at t12 PS80 at t0
PS80 at t12
Impact of Polysorbate degradation on protein formulationsSurface pressure in aged formulations
Insoluble degradants
PS20 PS80
0
0.1
0.2
0.3
0.4
0.5
F1 F3 F5 F7 F2 F4 F6 F8 F9 F11 F13 F15 F10 F12 F14 F16 F17 F18 F19 F20 F21 F22 F23 F24
mg/
ml S
urfa
ctan
t
t=9M5°C
t=9M25°C
PS20 PS80
0
5
10
15
20
25
30
35
40
45
%ox
idiz
ed
Impact of Polysorbate degradation on protein formulationsOxidation
+ Exc.X + Exc.X
+ Exc.X + Exc.X
Formulations F1-F24
Formulations F1-F24
Initial conc.
Surfactant content determination
% Fc Methionine Oxidation observed in protein at 25 °C
Impact of Polysorbate degradation on protein formulationsSummary
• Surface pressure experiments show comparable surface tension
• End of shelf life studies show effective protection even with decreased surfactant content
• Surface pressure experiments show comparable surface tension
• End of shelf life studies show effective protection even with decreased surfactant content
Effect of decreased surfactant content
• Fatty acids can appear as visible or subvisibleparticles
• Fatty acids may induce aggregation, above a threshold concentration
• Fatty acids can appear as visible or subvisibleparticles
• Fatty acids may induce aggregation, above a threshold concentration
Effect of insoluble degradants from
hydrolysis
• Degradation of polysorbates correlates to extent of oxidation in mAbs
• It is possible that peroxides generated from PS degradation causes mAb oxidation
• Degradation of polysorbates correlates to extent of oxidation in mAbs
• It is possible that peroxides generated from PS degradation causes mAb oxidation
Effect of peroxides from auto-oxidation
Overall Summary and Conclusions
• Polysorbates are a complex mixture of Sorbitan-POE-fattyacid esters
• Polysorbates stabilize (most) Proteins (against interfacial stress-induced aggregation) and can minimize protein adsorption to interfaces
• Polysorbates can adsorb to e.g. filters and other material
• Degradation may occur in bulk and/or pharmaceutical formulations via hydrolysis or by auto-oxidation (major pathway)
• Polysorbates can degrade and this requires sufficient attention during formulation development
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