Slide 1 A New Look at Old Stuff. Molecular Heterogeneity of Polysorbates and …c.ymcdn.com/sites/casss.site-ym.com/resource/resmgr/… · · 2014-07-21Molecular Heterogeneity of

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 5 Molecular Complexity of Polysorbates.

Slide 6 Molecular Complexity of Polysorbates.

http://www.rivalart.com/cart/pc/showsearchresults.asp?pageStyle=H&resultCnt=18&keyword=water&submit.x=0&submit.y=0&submit=submit

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


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


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


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






40 – 60%Lauric (C12)



less 1%Caproic (C6)


280.24

282.26

284.27

256.24

228.21

200.18

172.15

144.12

116.08

MW, Da






40 – 60%Lauric (C12)



less 1%Caproic (C6)


280.24

282.26

284.27

256.24

228.21

200.18

172.15

144.12

116.08

MW, Da






40 – 60%Lauric (C12)



less 1%Caproic (C6)


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 29 Different Esters Show Different Kinetics of

Oxidation.

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.

Slide 34 Acknowledgments

Melissa Alvarez

Dan Hewitt

John Wang

Victor Ling

Andrea Ji

Dan Zarraga

Felix Vega

Bruce Kerwin

Jia Yao

Documents

Slide 1 A New Look at Old Stuff. Molecular Heterogeneity of Polysorbates and …c.ymcdn.com/sites/casss.site-ym.com/resource/resmgr/… · · 2014-07-21Molecular Heterogeneity of