slide 1 a new look at old stuff. molecular heterogeneity of polysorbates and...
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Slide 1
A New Look at Old Stuff.
Molecular Heterogeneity of Polysorbates
and Its Implications Studied with LC-MS.
Oleg Borisov
9th Symposium on the Practical Applications of Mass
Spectrometry in the Biotechnology Industry
09/14/12
Slide 2 Properties of Polysorbates
• Non-Ionic Amphiphilic Surfactants (HLB > 10, O/W)
hydrophilic head
hydrophobic tail
• Trade names: Tween, Crillet, Sorlate, Monitan, Olothorb…
• General: Emulsifiers and stabilizers in foods, cosmetics,
drugs, textiles, plastics, agricultural chemicals,
• Biothech: Minimize protein adsorption to surfaces and to
reduce the air-liquid and solid-liquid interfacial surface
tension (aggregation). Stabilizing agent.
Slide 3
x + y + z + w = 20
“Mixture of partial esters of fatty acids, mainly lauric
acid, with sorbitol and its anhydrides ethoxylated with
approximately 20 moles of ethhylene oxide for each
mole of sorbitol and sorbitol anhydrides.”
For example, PS20 is described as:
What Is Polysorbate?
O
O(CH2CH2O)xHHw(OCH2CH2)O O(CH2CH2O)yH
O(CH2CH2O)x R
O
(USP-NF and EU Pharmacopoeia)
Slide 4
According to European Pharmacopoeia 6.3
Fatty Acid Structure MW, Da Fatty Acid Content, %
Polysorbate 20 Polysorbate 80
Caproic (C6) CH3(CH2)4COOH 116.08 < 1% ---
Caprylic (C8) CH3(CH2)6COOH 144.12 < 10% ---
Capric (C10) CH3(CH2)8COOH 172.15 < 10% ---
Lauric (C12) CH3(CH2)10COOH 200.18 40 – 60% ---
Myristic (C14) CH3(CH2)12COOH 228.21 14 – 25% < 5%
Palmitic (C16) CH3(CH2)14COOH 256.24 7 – 15% < 16%
Palmitoleic (C16:1) CH3(CH2)5CH=CH(CH2)7COOH 254.22 --- < 8%
Stearic (C18) CH3(CH2)16COOH 284.27 < 7% > 6%
Oleic (C18:1) CH3(CH2)7CH=CH(CH2)7COOH 282.26 < 11% > 58%
Linoleic (C18:2) CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH 280.24 < 3% < 18%
Linolenic (C18:3) CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH 278.22 --- < 4%
Heterogeneity with Regard to FAs
Slide 6 Molecular Complexity of Polysorbates.
Slide 7
• What do we know about Polysorbates?
• A look back,
• A novel LC-MS method to study Polysorbates
• Degradation of Polysorbates
• Studying and monitoring degradation by LC-MS
Outline
Slide 8
• Ethoxylation of fatty acids for making non-ionic surfactants 1928
• Solubilization of hydrophobic fatty acids with POE by Schöller 1930
• Process for Preparing Sorbitan Esters, US Patent by Stockburger 1981
Background. History of Ethoxylation.
• The oxyethylation reaction under basic conditions promotes ester
interchange resulting in random addition of EO to the hydroxyls.
• The total chain length (w + x + y + x) averages 20 units
• Some ethoxylated sorbitan molecules will also contain 0 or 2 or more
fatty acids per molecule,
• Anhydrization of sorbitol produces a mixture of 1,4-sorbitan and
isosorbide.
Slide 9
G.J. Stockburger. “Ethoxylation”. J. Am. Oil Chemists’ Soc., November 1979 (VOL. 56), 774A-777A.
Ethoxylation of Fatty Acids:
Simple Reaction – Multiple Products.
+
Slide 10
Sorbitol Mono-Anhydrides Sorbitol Di-Anhydrides
Molecular Heterogeneity of Polysorbates.
Beyond Diversity of Fatty Acids.
Other Polyols
May be present as
• Polyols,
• Mono-,
• Di-
• Tri-,
• Tetra-esters
May be present as
• Polyols,
• Mono-,
• Di-esters
May be present as
• Polyols,
• Mono-,
• Di-esters
Slide 11
• Composition of Polysorbates described by Brandner 1998
indicated that PS are esters of sorbitol mono- and di-anhydrides,
mono-, di-, and tri-esters are the most abundant compounds,
more than 20 moles of EO are combined.
History of Ethoxylation. Continued.
John D. Brandner. “The Composition of NF-Defined Emulsifiers: Sorbitan Monolaurate, Monopalmitate,
Monostearate, Monooleate, Polysorbate 20, Polysorbate 40, Polysorbate 60, and Polysorbate 80”.
Drug Development and Industrial Pharmacy, 24 ( 11), 1049-1054 (1998).
Slide 12 LC-MS Analysis of Polysorbates
Group 1
Group 2
Group 3
1: TOF MS ES+ TIC
2.27e5
1: TOF MS ES+ TIC
7.87e4
Time10.00 20.00 30.00 40.00 50.00 60.00
%
0
100
10.00 20.00 30.00 40.00 50.00 60.00
%
0
10031.6
26.9
54.532.9
41.233.8
35. 6 43.3 45.4
55.4
36.5
48.9
38.7
PS20
PS80
Slide 13
10.00 20.00 30.00 40.00 50.00 60.00
%
0
10031.6
26.9
54.532.9
41.233.8
35. 6 43.3 45.4
PS20
%
0
100
23*
28*
25*(M+2Na)2+
(17 – 38)
(15 – 34)
(M+Na)+
(23 – 33)
(M+3Na)3+
POE Sorbitan Mono-Laurate
LC-MS Analysis of Polysorbates. PS 20.
Slide 14
10.00 20.00 30.00 40.00 50.00 60.00
%
0
10031.6
26.9
54.532.9
41.233.8
35. 6 43.3 45.4
PS20
%
0
100
11*
(M+Na)+
(6 – 21)14*
(10 – 22)
(M+2Na)2+
POE Isosorbide Mono-Laurate
LC-MS Analysis of Polysorbates. PS 20.
Slide 15
10.00 20.00 30.00 40.00 50.00 60.00
%
0
10031.6
26.9
54.532.9
41.233.8
35. 6 43.3 45.4
PS20
m/z400 600 800 1000 1200 1400 1600
%
0
100
12*
(M+Na)+
(7 – 19)
14*
(11 – 19)
(M+2Na)2+
POE Mono-Laurate
LC-MS Analysis of Polysorbates. PS 20.
Slide 16
10.00 20.00 30.00 40.00 50.00 60.00
%
0
10031.6
26.9
54.532.9
41.233.8
35. 6 43.3 45.4
PS20
m/z400 600 800 1000 1200 1400 1600
%
0
100
23*
28*
25*
(17 – 38)
(18 – 30)
(23 – 34)
(M+3Na)3+
(M+Na)+
(M+2Na)2+
POE Sorbitan Di-Laurate
LC-MS Analysis of Polysorbates. PS 20.
Slide 17 LC-MS Analysis of Polysorbates
A Typical Condition
“In-Source” CID or “who messed
with my instrument?” Condition
Slide 18
m/z200 400 600 800 1000 1200 1400 1600 1800
%
0
100
%
0
100
%
0
100
(M+2Na)2+
(M+2Na)2+
(M+2Na)2+
Sorbitan POE laurate
Sorbitan POE di-laurate
Sorbitan POE laurate/myristate
m/z200 400 600 800 1000 1200 1400 1600 1800
%
0
100
%
0
100
%
0
100
(M+2Na)2+
(M+2Na)2+
(M+2Na)2+
Sorbitan POE laurate
Sorbitan POE di-laurate
Sorbitan POE laurate/myristate
CID of POE Sorbitan Esters with 26 EO Units.
(M+Na)+
(M+Na)+
(M+Na)+
3EO
3EO
3EO
3EO
3EO
3EO
O
HO
+ O
HO
+
2
O
HO
+ O
HO
+
2
O
HO
+ O
HO
+
2
25
5.2
3
22
7.2
0
22
7.2
0
22
7.2
0
Slide 19
n
CID
Possible Mechanism of 1,3-Dioxolanylium Ion
Formation.
n Molecular Weight:
FA + C2H3 (27 Da)
• Fragmentation of sodiated precursors produces abundant dioxolanylium ions,
characteristic to Fatty Acid component.
Slide 20 Profiling Fatty Acids in Polysorbate 20.
Time, min
10 20 30 40 50 60
Sig
nal
0
200
400
600
800
1000
1200
1400
Caprylic C8:0
Capric C10:0
Lauric C12:0
Myristic C14:0
Palmitic C16:0
Stearic C18:0
Oleic C18:1
1
2
3 4
5
6 7 8
9
1 – POE Sorbitan Laurate;
2 – POE Isosorbide Laurate;
3-7 – POE Sorbitan Di-esters;
8 – POE Sorbitan Tri-ester;
9 – POE Sorbitan Tetra-ester.
m/z
Slide 21 Profiling Fatty Acids in Polysorbate 20.
280.24
282.26
284.27
256.24
228.21
200.18
172.15
144.12
116.08
MW, Da
less 3%Linoleic (C18 2 unsat.)
less 11%Oleic (C18 1 unsat.)
less 7%Stearic (C18)
7 – 15%Palmitic (C16)
14 – 25%Myristic (C14)
40 – 60%Lauric (C12)
less 10%Capric (C10)
less 10%Caprylic (C8)
less 1%Caproic (C6)
Expected, %Fatty Acid
280.24
282.26
284.27
256.24
228.21
200.18
172.15
144.12
116.08
MW, Da
less 3%Linoleic (C18 2 unsat.)
less 11%Oleic (C18 1 unsat.)
less 7%Stearic (C18)
7 – 15%Palmitic (C16)
14 – 25%Myristic (C14)
40 – 60%Lauric (C12)
less 10%Capric (C10)
less 10%Caprylic (C8)
less 1%Caproic (C6)
Expected, %Fatty Acid
280.24
282.26
284.27
256.24
228.21
200.18
172.15
144.12
116.08
MW, Da
less 3%Linoleic (C18 2 unsat.)
less 11%Oleic (C18 1 unsat.)
less 7%Stearic (C18)
7 – 15%Palmitic (C16)
14 – 25%Myristic (C14)
40 – 60%Lauric (C12)
less 10%Capric (C10)
less 10%Caprylic (C8)
less 1%Caproic (C6)
Expected, %Fatty Acid
280.24
282.26
284.27
256.24
228.21
200.18
172.15
144.12
116.08
MW, Da
less 3%Linoleic (C18 2 unsat.)
less 11%Oleic (C18 1 unsat.)
less 7%Stearic (C18)
7 – 15%Palmitic (C16)
14 – 25%Myristic (C14)
40 – 60%Lauric (C12)
less 10%Capric (C10)
less 10%Caprylic (C8)
less 1%Caproic (C6)
Expected, %Fatty Acid
Slide 22
TIC
0.0
2.5e+4
5.0e+4
7.5e+4
Time, min
5 15 25 35 45 55
Sig
na
l
0
500
1000
1500
2000
2500
Fatty Acid Lipid Number Relative Amount, %
Myristic C14:0 2.9
Palmitic C16:0 5.8
Palmitoleic C16:1 6.8
Stearic C18:0 2.0
Oleic C18:1 77.0
Linoleic C18:2 3.6
Linolenic C18:3 1.9
1 2 3 4 5
13.6 15.8
36.6
38.9
48.9
55.6
36.6
38.9 48.9
51.8
55.5
Myristic
Palmitic
Palmitoleic
Stearic
Oleic
Linoleic
Linolenic
Profiling Fatty Acids in Polysorbate 80.
polyols
mono-oleates
di-oleates
tri-oleates
Slide 23
Method
POE Sorbitan Esters
Mono- Di- Tri-
LC-MS (RIC m/z 227) 43 37 20
Brandner (1998)* 49 38 13
* John D. Brandner. “The Composition of NF-Defined Emulsifiers: Sorbitan Monolaurate, Monopalmitate,
Monostearate, Monooleate, Polysorbate 20, Polysorbate 40, Polysorbate 60, and Polysorbate 80”. Drug
Development and Industrial Pharmacy, 24 ( 11), 1049-1054 (1998).
Profiling Polysorbate 20 with LC-MS.
Slide 24 Stability of Polysorbates in Relevance to
Bioterapeutics.
• E. Ha et al. “Peroxide formation in polysorbate 80 and protein stability.” J Pharm
Sci. 2002, 91, 2252-2264.
• W. Wang et al. “Dual effects of Tween 80 on protein stability.” Int. J. Pharm. 2008,
347, 31-38.
• B. Kerwin “Polysorbates 20 and 80 used in the formulation of protein
biotherapeutics: structure and degradation pathways.” J. Pharm. Sci. 2008, 97,
2924-2935.
• J. Yao et al. “A quantitative kinetic study of polysorbate autoxidation: the role
of unsaturated fatty acid ester substituents.” Pharm. Res. 2009, 26, 2303-2313.
• D. Hewitt et al. “Mixed-mode and reversed-phase liquid chromatography-
tandem mass spectrometry methodologies to study composition and base
hydrolysis of polysorbate 20 and 80” J. Chromatogr. A 2011,1218, 2138-2145.
• R. Kishore et al. “The Degradation of Polysorbates 20 and 80 and its Potential
Impact on the Stability of Biotherapeutics” Pharm. Res. 2011, 28, 1194-1210.
Slide 25
• Degradation of polysorbates, role of autoxidation 1978
Stability of Polysorbates. Autoxidation.
Donbrow, M., et al. “Autoxidation of Polysorbates.” J. Pharm. Sci. 1978, 67, 1676-1681.
Slide 26
• R. Kishore et al. “The Degradation of Polysorbates 20 and 80 and its Potential Impact
on the Stability of Biotherapeutics” Pharm. Res. 2011, 28, 1194-1210.
• Peroxides,
• Short chain organic acids,
• Aldehydes,
• Ketones,
• N-alkanes,
• Fatty acid esters
• Fatty acids,
• POE sorbitans
Stability of Polysorbates.
Autoxidation
(oxidizers, light, metals) Hydrolysis
(pH)
Slide 27 Oxidation of PS 20. What to Expect?
POE Chain Shortening POE Ester
POE Sorbitan
Using AAPH to study oxidation of polysorbates
2,2’-azobis(amidinopropane) dihydrochloride
Slide 28
EO Number
EO Number
POE
Mono-Laurate
POE Sorbitan
Mono-Laurate
Oxidation of PS 20 with AAPH.
Slide 30
AAPH
POE oleates
Hydropeoxy-,
Hydroxy-,
Epoxy-,
Oxo-Nonanoates
n
C8H17 (CH2)7 O
O
CH2CH2O
n 9
Corresponding POE mono-esters
I II
IV III
Pathways of Oxidative Degradation of PS80.
127
133
0
25
50
75
100
125
150
175
200
0 500 1000 1500 2000 2500
Pe
ak
Are
a
Time, min
POE (26) sorbitan oleate
Path I (POE (26) sorbitan esters)
Path II (POE (5) oleate)
Path I & II (POE (5) esters)
Slide 31
Epoxy-Octadecanoate
Oxo-Nonanoate
Hydroxy-Octadecenoate
Oxidation of PS 80 with AAPH.
0
2
4
6
8
10
12
14
16
18
20
11 21 31 41 51
x 1
00
00
T0
2.5 h
6.3 h
12.5 h
18.8 h
1.5 mM AAPH
Slide 32
Mono-Laurate
Mono-C18
Di-Laurate Mono-Laurate
Di-Laurate
Mono-C18:1
Mono-C18:0
Degradation of PS 20.
Oxidation Hydrolysis* versus
Time
32.5
15.227.7
55.5542.1
38.9
10 15 20 25 30 35 40 45 50 55 60 65
%
0
100 Oxidation Hydrolysis
* D. Hewitt et al. J. Chromatogr. A 1218 (2011) 2138–2145.
5mM AAPH
Slide 33 Conclusions
• Polysorbates are heterogeneous. No doubt
about that.
• Quite possibly that what makes them good surfactants
• LC-MS offers (and dioxalanilyum ions can help with):
• Distribution of Fatty Acids and other constituents,
• Monitoring stability of polysorbates,
• Detecting and identifying degradation products and
their pathways,
• Telling what happened to your polysorbate before you
got to it.