A New Look at Old Stuff. Molecular Heterogeneity of Polysorbates and Its Implications Studied with LC-MS.

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1 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 9/14/12

2 Properties of Polysorbates Slide 2 Non-Ionic Amphiphilic Surfactants (HLB > 1, 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.

3 What Is Polysorbate? Slide 3 For example, PS2 is described as: Mixture of partial esters of fatty acids, mainly lauric acid, with sorbitol and its anhydrides ethoxylated with approximately 2 moles of ethhylene oxide for each mole of sorbitol and sorbitol anhydrides. (USP-NF and EU Pharmacopoeia) H w (OCH 2 CH 2 )O O(CH 2 CH 2 O) x H O(CH 2 CH 2 O) y H O O(CH 2 CH 2 O) x R x + y + z + w = 2 O

4 Heterogeneity with Regard to FAs Slide 4 Fatty Acid Structure MW, Da Fatty Acid Content, % Polysorbate 2 Polysorbate 8 Caproic (C6) CH 3 (CH 2 ) 4 COOH < 1% --- Caprylic (C8) CH 3 (CH 2 ) 6 COOH < 1% --- Capric (C1) CH 3 (CH 2 ) 8 COOH < 1% --- Lauric (C12) CH 3 (CH 2 ) 1 COOH % --- Myristic (C14) CH 3 (CH 2 ) 12 COOH % < 5% Palmitic (C16) CH 3 (CH 2 ) 14 COOH % < 16% Palmitoleic (C16:1) CH 3 (CH 2 ) 5 CH=CH(CH 2 ) 7 COOH < 8% Stearic (C18) CH 3 (CH 2 ) 16 COOH < 7% > 6% Oleic (C18:1) CH 3 (CH 2 ) 7 CH=CH(CH 2 ) 7 COOH < 11% > 58% Linoleic (C18:2) CH 3 (CH 2 ) 4 CH=CHCH 2 CH=CH(CH 2 ) 7 COOH < 3% < 18% Linolenic (C18:3) CH 3 CH 2 CH=CHCH 2 CH=CHCH 2 CH=CH(CH 2 ) 7 COOH < 4% According to European Pharmacopoeia 6.3

5 Molecular Complexity of Polysorbates. Slide 5

6 Molecular Complexity of Polysorbates. Slide 6

7 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

8 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 193 Process for Preparing Sorbitan 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 2 units Some ethoxylated sorbitan molecules will also contain or 2 or more fatty acids per molecule, Anhydrization of sorbitol produces a mixture of 1,4-sorbitan and isosorbide.

9 Ethoxylation of Fatty Acids: Simple Reaction Multiple Products. Slide 9 + G.J. Stockburger. Ethoxylation. J. Am. Oil Chemists Soc., November 1979 (VOL. 56), 774A-777A.

10 Molecular Heterogeneity of Polysorbates. Beyond Diversity of Fatty Acids. Slide 1 Sorbitol Mono-Anhydrides Sorbitol Di-Anhydrides May be present as Polyols, Mono-, Di- Tri-, Tetra-esters Other Polyols May be present as Polyols, Mono-, Di-esters May be present as Polyols, Mono-, Di-esters

11 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 2 moles of EO are combined. John D. Brandner. The Composition of NF-Defined Emulsifiers: Sorbitan Monolaurate, Monopalmitate, Monostearate, Monooleate, Polysorbate 2, Polysorbate 4, Polysorbate 6, and Polysorbate 8. Drug Development and Industrial Pharmacy, 24 ( 11), (1998).

12 % % LC-MS Analysis of Polysorbates Slide 12 1 PS2 Group Group : TOF MS ES+ TIC 2.27e5 Group PS : TOF MS ES+ TIC 7.87e Time

13 % % LC-MS Analysis of Polysorbates. PS 2. Slide 13 1 PS (17 38) 25* (M+2Na) 2+ (M+3Na) 3+ (23 33) 28* (M+Na) + (15 34) 23* POE Sorbitan Mono-Laurate

14 % % LC-MS Analysis of Polysorbates. PS 2. Slide 14 1 PS (M+2Na) 2+ (1 22) 14* (M+Na) + (6 21) 11* POE Isosorbide Mono-Laurate

15 % % LC-MS Analysis of Polysorbates. PS 2. Slide 15 1 PS (M+2Na) 2+ (11 19) 14* (7 19) 12* (M+Na) POE Mono-Laurate m/z

16 % % LC-MS Analysis of Polysorbates. PS 2. Slide 16 1 PS (17 38) 25* (M+2Na) 2+ (M+3Na) 3+ (23 34) 28* (M+Na) + (18 3) 23* POE Sorbitan Di-Laurate m/z

17 LC-MS Analysis of Polysorbates Slide 17 A Typical Condition In-Source CID or who messed with my instrument? Condition

18 % % % CID of POE Sorbitan Esters with 26 EO Units. Slide 18 Sorbitan POE laurate 1 (M+Na) + HO 1 HO O + O + (M+2Na) 2+ 3EO (M+2Na) 2+ 3EO Sorbitan POE di-laurate (M+Na) + HO O HO 1 + O + 3EO (M+2Na) 2+ 3EO Sorbitan POE laurate/myristate (M+Na) + HO O HO + O + 3EO 3EO m/z

19 Possible Mechanism of 1,3-Dioxolanylium Ion Formation. Slide 19 CID n n Molecular Weight: FA + C 2 H 3 (27 Da) Fragmentation of sodiated precursors produces abundant dioxolanylium ions, characteristic to Fatty Acid component.

20 m/z Profiling Fatty Acids in Polysorbate 2. Slide 2 Stearic C18: Oleic C18:1 Palmitic C16: Myristic C14: Lauric C12: Capric C1: Caprylic C8: Signal POE Sorbitan Laurate; 2 POE Isosorbide Laurate; 3-7 POE Sorbitan Di-esters; 8 POE Sorbitan Tri-ester; 9 POE Sorbitan Tetra-ester Time, min

21 Profiling Fatty Acids in Polysorbate 2. Slide 21 Fatty Acid Fatty Acid MW, Da Caproic (C6) Caproic (C6) Caprylic (C8) Caprylic (C8) Capric (C1) Capric (C1) Lauric (C12) Lauric (C12) 2.18 Myristic (C14) Myristic (C14) Palmitic (C16) Palmitic (C16) Stearic (C18) Stearic (C18) Oleic (C18 1 Oleic unsat.) (C18 1 unsat.) Linoleic (C18 Linoleic 2 unsat.) (C unsat.) MW, Expected, Da % Expected, less 1% less 1% less 1% % % % less 7% less 11% less 3% less 1% less 1% less 1% 4 6% 14 25% 7 15% less 7% less 11% less 3%

22 Profiling Fatty Acids in Polysorbate e Slide 22 TIC 5.e+4 2.5e+4 polyols Fatty Acid Lipid Number Relative Amount, % Myristic C14: 2.9 Palmitic C16: 5.8 Palmitoleic C16:1 6.8 Stearic C18: 2. Oleic C18:1 77. Linoleic C18:2 3.6 Linolenic C18: Myristic Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic Signal mono-oleates di-oleates 48.9 tri-oleates Time, min

23 Profiling Polysorbate 2 with LC-MS. Slide 23 Method POE Sorbitan Esters Mono- Di- Tri- LC-MS (RIC m/z 227) Brandner (1998)* * John D. Brandner. The Composition of NF-Defined Emulsifiers: Sorbitan Monolaurate, Monopalmitate, Monostearate, Monooleate, Polysorbate 2, Polysorbate 4, Polysorbate 6, and Polysorbate 8. Drug Development and Industrial Pharmacy, 24 ( 11), (1998).

24 Stability of Polysorbates in Relevance to Bioterapeutics. Slide 24 E. Ha et al. Peroxide formation in polysorbate 8 and protein stability. J Pharm Sci. 22, 91, W. Wang et al. Dual effects of Tween 8 on protein stability. Int. J. Pharm. 28, 347, B. Kerwin Polysorbates 2 and 8 used in the formulation of protein biotherapeutics: structure and degradation pathways. J. Pharm. Sci. 28, 97, J. Yao et al. A quantitative kinetic study of polysorbate autoxidation: the role of unsaturated fatty acid ester substituents. Pharm. Res. 29, 26, D. Hewitt et al. Mixed-mode and reversed-phase liquid chromatographytandem mass spectrometry methodologies to study composition and base hydrolysis of polysorbate 2 and 8 J. Chromatogr. A 211,1218, R. Kishore et al. The Degradation of Polysorbates 2 and 8 and its Potential Impact on the Stability of Biotherapeutics Pharm. Res. 211, 28,

25 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,

26 Stability of Polysorbates. Slide 26 Autoxidation (oxidizers, light, metals) Hydrolysis (ph) Peroxides, Short chain organic acids, Aldehydes, Ketones, N-alkanes, Fatty acid esters Fatty acids, POE sorbitans R. Kishore et al. The Degradation of Polysorbates 2 and 8 and its Potential Impact on the Stability of Biotherapeutics Pharm. Res. 211, 28,

27 Oxidation of PS 2. What to Expect? Slide 27 POE Sorbitan POE Chain Shortening POE Ester Using AAPH to study oxidation of polysorbates 2,2 -azobis(amidinopropane) dihydrochloride

28 Oxidation of PS 2 with AAPH. Slide 28 POE Sorbitan Mono-Laurate EO Number POE Mono-Laurate EO Number

29 Different Esters Show Different Kinetics of Oxidation. Slide 29

30 Peak Area Pathways of Oxidative Degradation of PS8. Slide 3 C 8 H 17 (CH 2 ) 7 O 9 O AAPH CH 2 CH 2 O n POE (26) sorbitan oleate Path I (POE (26) sorbitan esters) Path II (POE (5) oleate) n 15 Path I & II (POE (5) esters) I II Hydropeoxy-, Hydroxy-, Epoxy-, Oxo-Nonanoates POE oleates 75 5 III IV 25 Corresponding POE mono-esters Time, min

31 x 1 Oxidation of PS 8 with AAPH. Slide 31 Oxo-Nonanoate Hydroxy-Octadecenoate Epoxy-Octadecanoate T 2.5 h 6.3 h 12.5 h 18.8 h 1.5 mm AAPH

32 % Degradation of PS 2. Slide 32 Oxidation 5mM AAPH Mono-Laurate Mono-C18: Di-Laurate Mono-C18:1 versus Hydrolysis* Di-Laurate Mono-C18 Mono-Laurate 1 Hydrolysis Oxidation Time * D. Hewitt et al. J. Chromatogr. A 1218 (211)

33 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.

34 Acknowledgments Slide 34 Melissa Alvarez Dan Hewitt John Wang Victor Ling Andrea Ji Dan Zarraga Felix Vega Bruce Kerwin Jia Yao