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 Properties of Polysorbates Slide 2

• 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. What Is Polysorbate? Slide 3

For example, PS20 is described as:

“Mixture of partial esters of fatty acids, mainly , with and its anhydrides ethoxylated with approximately 20 moles of ethhylene oxide for each mole of sorbitol and sorbitol anhydrides.”

(USP-NF and EU Pharmacopoeia)

O(CH2CH2O)xH Hw(OCH2CH2)O O(CH2CH2O)yH

O O(CH2CH2O)x R x + y + z + w = 20 O Heterogeneity with Regard to FAs Slide 4

Fatty Acid Content, % Fatty Acid Structure MW, Da 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%

According to European Pharmacopoeia 6.3 Molecular Complexity of Polysorbates. Slide 5 Molecular Complexity of Polysorbates. Slide 6 Outline 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 Background. History of Ethoxylation. 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 Esters, US Patent by Stockburger 1981

• 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 . Ethoxylation of Fatty Acids: Slide 9 Simple Reaction – Multiple Products.

+

G.J. Stockburger. “Ethoxylation”. J. Am. Oil Chemists’ Soc., November 1979 (VOL. 56), 774A-777A. Molecular Heterogeneity of Polysorbates. Beyond Diversity of Fatty Acids. Slide 10

Sorbitol Mono-Anhydrides Sorbitol Di-Anhydrides

May be present as May be present as • Polyols, • Polyols, • Mono-, • Mono-, • Di- • Di-esters • Tri-, Other Polyols • Tetra-esters

May be present as • Polyols, • Mono-, • Di-esters History of Ethoxylation. Continued. 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.

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). LC-MS Analysis of Polysorbates Slide 12

31.6 100 Group 2 PS20 1: TOF MS ES+ TIC 54.5 2.27e5 32.9 Group 3 Group 1 % 33.8 41.2

35. 6 43.3 45.4 26.9

0 10.00 20.00 30.00 40.00 50.00 60.00

55.4 100 1: TOF MS ES+ PS80 TIC 7.87e4

36.5

48.9 % 38.7

0 Time 10.00 20.00 30.00 40.00 50.00 60.00 LC-MS Analysis of Polysorbates. PS 20. Slide 13

31.6 100 PS20 54.5 32.9

% 33.8 41.2

35. 6 43.3 45.4 26.9

0 10.00 20.00 30.00 40.00 50.00 60.00

100 (17 – 38) 25* (M+2Na)2+

(M+3Na)3+

% (23 – 33) 28* (M+Na)+ (15 – 34)

23*

0 POE Sorbitan Mono-Laurate LC-MS Analysis of Polysorbates. PS 20. Slide 14

31.6 100 PS20 54.5 32.9

% 33.8 41.2

35. 6 43.3 45.4 26.9

0 10.00 20.00 30.00 40.00 50.00 60.00

(M+2Na)2+ 100 (10 – 22) (M+Na)+ 14* (6 – 21)

11* %

0 POE Isosorbide Mono-Laurate LC-MS Analysis of Polysorbates. PS 20. Slide 15

31.6 100 PS20 54.5 32.9

% 33.8 41.2

35. 6 43.3 45.4 26.9

0 10.00 20.00 30.00 40.00 50.00 60.00

100 (7 – 19) 12* (M+Na)+ (M+2Na)2+ (11 – 19)

% 14*

0 m/z 400 600 800 1000 1200 1400 1600 POE Mono-Laurate LC-MS Analysis of Polysorbates. PS 20. Slide 16

31.6 100 PS20 54.5 32.9

% 33.8 41.2

35. 6 43.3 45.4 26.9

0 10.00 20.00 30.00 40.00 50.00 60.00

100 (17 – 38)

25* (M+2Na)2+

(M+3Na)3+ % (23 – 34) + 28* (M+Na) (18 – 30)

23* 0 m/z 400 600 800 1000 1200 1400 1600 POE Sorbitan Di-Laurate LC-MS Analysis of Polysorbates Slide 17

A Typical Condition

“In-Source” CID or “who messed with my instrument?” Condition

CID of POE Sorbitan Esters with 26 EO Units.Slide 18

Sorbitan POE laurate (M+Na)+ 100

HO

227.20

HO 2

O % % (M+2Na)2+ O + 3EO + 3EO

0 Sorbitan POE di-laurate (M+Na)+

100 227.20 (M+2Na)2+

2

% % + 3EO + 3EO 0 Sorbitan POE laurate/myristate (M+2Na)2+

+

100 (M+Na)

2

227.20 255.23

% % + 3EO 3EO + 0 m/z 200 400 600 800 1000 1200 1400 1600 1800 Possible Mechanism of 1,3-Dioxolanylium Ion Slide 19 Formation.

CID n

n Molecular Weight:

FA + C2H3 (27 Da)

• Fragmentation of sodiated precursors produces abundant dioxolanylium ions, characteristic to Fatty Acid component. Profiling Fatty Acids in Polysorbate 20. Slide 20

Stearic C18:0

Oleic C18:1

Palmitic C16:0

Myristic C14:0 m/z Lauric C12:0

Capric C10:0

Caprylic C8:0

1400 1200 1 – POE Sorbitan Laurate; 1 1000 2 – POE Isosorbide Laurate; 800 3-7 – POE Sorbitan Di-esters; 5 2 8 – POE Sorbitan Tri-ester; 600 9 Signal 400 9 – POE Sorbitan Tetra-ester. 6 7 8 200 3 4 0

10 20 30 40 50 60 Time, min Profiling Fatty Acids in Polysorbate 20. Slide 21

Fatty Acid Fatty AcidMW, Da MW,Expected, Da %Expected, % Caproic (C6)Caproic (C6)116.08 116.08less 1% less 1% Caprylic (C8)Caprylic (C8)144.12 144.12less 10% less 10% Capric (C10)Capric (C10)172.15 172.15less 10% less 10% Lauric (C12)Lauric (C12)200.18 200.1840 – 60% 40 – 60% Myristic (C14)Myristic (C14)228.21 228.2114 – 25% 14 – 25% Palmitic (C16)Palmitic (C16)256.24 256.247 – 15% 7 – 15% Stearic (C18)Stearic (C18)284.27 284.27less 7% less 7% Oleic (C18 1Oleic unsat (C18.) 1 unsat282.26.) 282.26less 11% less 11% Linoleic (C18Linoleic 2 unsat(C18.) 2 280.24unsat.) 280.24less 3% less 3% 7.5e+4Profiling Fatty Acids in Polysorbate 80. 55.6 Slide 22

36.6 5.0e+4 48.9

TIC 38.9 polyols 2.5e+4 13.6 15.8

0.0 Myristic Fatty Acid Lipid Number Relative Amount, % Myristic C14:0 2.9 Palmitic Palmitic C16:0 5.8 Palmitoleic Palmitoleic C16:1 6.8 Stearic C18:0 2.0 Stearic Oleic C18:1 77.0 1 2 3 4 5 Linoleic C18:2 3.6 Oleic Linolenic C18:3 1.9 Linoleic

Linolenic

mono-oleates 2500 36.6 di-oleates 2000 38.9 48.9 tri-oleates 1500 55.5

1000 Signal 51.8

500

0 5 15 25 35 45 55 Time, min Profiling Polysorbate 20 with LC-MS. Slide 23

POE Sorbitan Esters Method 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). Stability of Polysorbates in Relevance to Slide 24 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.

Stability of Polysorbates. Autoxidation. Slide 25

• Degradation of polysorbates, role of autoxidation 1978

Donbrow, M., et al. “Autoxidation of Polysorbates.” J. Pharm. Sci. 1978, 67, 1676-1681. Stability of Polysorbates. Slide 26

Autoxidation (oxidizers, light, metals) Hydrolysis (pH)

• Peroxides, • Short chain organic acids, • Aldehydes, • Fatty acids, • Ketones, • POE sorbitans • N-alkanes, • Fatty acid esters

• 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. Oxidation of PS 20. What to Expect? Slide 27

POE Sorbitan

POE Chain Shortening POE Ester

Using AAPH to study oxidation of polysorbates

2,2’-azobis(amidinopropane) dihydrochloride Oxidation of PS 20 with AAPH. Slide 28

POE Sorbitan Mono-Laurate

EO Number

POE Mono-Laurate

EO Number Different Esters Show Different Kinetics of Slide 29 Oxidation. Pathways of Oxidative Degradation of PS80. Slide 30

O POE (26) sorbitan oleate 200 C8H17 (CH2)7 O CH2CH2O 9 n AAPH Path I (POE (26) sorbitan esters) 175 Path II (POE (5) oleate)

150 Path I & II (POE (5) esters) n

133

127 I II 125

100 Peak Area Peak Hydropeoxy-, Hydroxy-, POE oleates 75 Epoxy-, Oxo-Nonanoates 50

III IV 25

0 0 500 1000 1500 2000 2500 Corresponding POE mono-esters Time, min Oxidation of PS 80 with AAPH. Slide 31

Oxo-Nonanoate Hydroxy-Octadecenoate

20 Epoxy-Octadecanoate

18 x 10000 x

16

14

12 T0 10 2.5 h 8

6 6.3 h

4 12.5 h 2 18.8 h 0 11 21 31 41 51 1.5 mM AAPH Degradation of PS 20. Slide 32 Oxidation versus Hydrolysis* 5mM AAPH Mono-Laurate Di-Laurate

Mono-C18:0 Mono-C18 Di-Laurate Mono-Laurate Mono-C18:1

32.5 100 Hydrolysis Oxidation 55.55 42.1

% 15.2 27.7 38.9

0 Time 10 15 20 25 30 35 40 45 50 55 60 65

* D. Hewitt et al. J. Chromatogr. A 1218 (2011) 2138–2145. Conclusions Slide 33

• 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. Acknowledgments Slide 34

Melissa Alvarez Dan Hewitt John Wang Victor Ling Andrea Ji Dan Zarraga Felix Vega

Bruce Kerwin Jia Yao