Selecting Polysorbates:

Transcription

1 Critical Considerations In Selecting Polysorbates: Proteins and Polysorbate Stability R. Matthew Fesinmeyer Principal Scientist, Drug Product Formulation Technologies AAPS Annual Meeting, November 6 th, 2014

2 Polysorbates a common non-ionic surfactant in Biopharma The polysorbates (20 and 80) are the most commonly used surfactants in protein parenteral drugs. They are proven surfactants, both with respect to protecting proteins and patient safety. The excipient family is not without challenges. 2

3 The CMC is in an imperfect predictor of polysorbate target concentration. Surface Tension (mn/m) 50 CMC [PS] << CMC [PS] < CMC CMC Polysorbate 80 Polysorbate 20 PS80 concentrations in marketed products PS20 concentrations in marketed products 20 [PS] = CMC [PS] > CMC Polysorbate Concentration ( M) Adapted from Kerwin, J. Pharm. Sci.,

4 ur model of polysorbate structure is relatively simple. H ( C H 2 C H 2 ) w ( C H 2 C H 2 ) x H MW = 1228 amu ( C H 2 C H 2 ) y H ( C H 2 C H 2 ) Z ( C H 2 ) 1 0 C H 3 Polysorbate 20 (PS20) polyoxyethylene (PE) sorbitan monolaurate H ( C H 2 C H 2 ) w ( C H 2 C H 2 ) x H MW = 1310 amu ( C H 2 C H 2 ) y H ( C H 2 C H 2 ) Z ( C H 2 ) 7 ( C H 2 ) 7 C H 3 Polysorbate 80 (PS80) polyoxyethylene (PE) sorbitan monooleate (w + x + y + z) = 20 Kerwin, BA (2008) J Pharm Sci 97:

5 But the head group isn t consistent Sorbitan Isosorbides Adapted from Borisov, et al., Anal. Chem,

6 and the tail group isn t either (and that s a requirement). PS20 PS80 caproic acid 1% CH 3 (CH 2 ) 4 CH caprylic acid 10% CH 3 (CH 2 ) 6 CH capric acid 10% CH 3 (CH 2 ) 8 CH lauric acid % CH 3 (CH 2 ) 10 CH myristic acid % 5% CH 3 (CH 2 ) 12 CH palmitic acid % 16% CH 3 (CH 2 ) 14 CH palmitoleic acid 8% CH 3 (CH 2 ) 5 CH=CH(CH 2 ) 7 C 2 H stearic acid 7% 6% CH 3 (CH 2 ) 16 CH oleic acid 11% % CH 3 (CH 2 ) 7 CH=CH(CH 2 ) 7 CH linoleic acid 3% 18% CH 3 (CH 2 ) 3 (CH 2 CH=CH) 2 (CH 2 ) 7 CH linolenic acid 4% 4% CH 3 (CH 2 CH=CH) 3 (CH 2 ) 7 CH European Pharmacopoeia nline, 5th edition, Version 5.2, polysorbate :0426; polysorbate :

7 LC-MS characterization is demonstrative of the overall heterogeneity. The TIC (top) and fatty-acid based RIC (heat map) of PS20 graphically demonstrate the overall heterogeneity of the raw material. Adapted from Borisov, et al., Anal. Chem, 2011, 83, pp

8 The raw material is heterogeneous and then it degrades. By oxidation CH 2 CH 2 CH 2 CH 2 H (CH 2 CH 2 ) z - Initiation (CH 2 ) 10 CH 3 Propogation Heat, Near-UV Light, Chemical initiators, or transition metals (i.e. FeIII) Termination CH 2 CH 2 CH 2 CHH 2 CH 2 CH 2 CH 2 CHH + CH 2 CH 2 H + CH 2 CH 2 CH 2 CH 2 H HCH (formic acid) HH (hydrogen peroxide) H 2 C (formaldehyde) CH 2 CH 2 CH 2 CHH H R + H R + H CH 2 CH 2 CH 2 CH 2 H R + R + H 2 H 3 CCH (acetic acid) + other inactive products through bimolecular reactions + 8 Adapted from Kerwin, J. Pharm. Sci., 2008, 97, pp

9 and hydrolysis. CH 2 CH 2 CH 2 CH 2 H (CH 2 CH 2 ) z - (CH 2 ) 10 CH 3 Acid or base Hydrolysis H + (CH 2 CH 2 ) z H (CH 2 ) 10 CH 3 9 Adapted from Kerwin, J. Pharm. Sci., 2008, 97, pp

10 Justifying polysorbate inclusion The need to include a surfactant is often readily obvious. Freeze/thaw stability Material loss to surfaces Aggregation due to surface interactions. The idea that the polysorbate is creating particles is less so. 10

11 Particles observed during formulation work with mabx 150 mg/ml mabx in salt-free, isotonic solution at ph << pi, 0.010% polysorbate. The opalescence in PS20 was caused by discrete particles that dispersed in the solution when swirled. PS20 PS80 The particles are not visible based on a typical cut-off value. 11

12 When is a particle not a particle? Visible Particle bserved by a trained inspector under controlled conditions. Typical lower-limit particle size of ~100 µm. Sub-Visible Particle Quantified by HIAC. Focused on 10 µm and 25 µm particle sizes to meet USP guidelines. Additional interest in 2 µm and/or particle counts from alternative instruments (eg, MFI). 12

13 mabx particles were sensitive to %PS20 Contrast-enhanced visible-light images (1 month, 4 C) 0.002% 0.002% 0.010% 0.004% 0.006% 0.014% 0.006% 0.016% 0.016% 0.016% 0.008% particles are approximately 2-10 µm % 0.02%

14 HIAC sub-visible particle counts. Sub-visible particle counts were not reflective of the visual observations. mabx consistently returned very low 10 µm and 25 µm particle counts. The 2 µm counts also remained very low. 14

15 The particles could be isolated by filtration, visualized, and analyzed by IR Micrograph 100x Raman microscope Adapted from Cao, et al., J. Pharm. Sci., 2014, nline 15

16 Where are the fatty acids coming from? Polysorbate hydrolysis at 5 C. The data on polysorbate hydrolysis is becoming increasingly detailed and supports slow, chemically induced hydrolysis over time. Adapted from Yi Li, et al. Anal. Chem, 2014, 86 pp

17 Where are the fatty acids coming from? Polysorbate hydrolysis at 5 C. PE Sorbitan mono-laurate lipase PE Sorbitan + lauric acid Formulation with a mab results in rapid hydrolysis. Consistent with the the presence of active lipase. To-date, a lipase has not been identified. Adapted from Labrenz, J. Pharm. Sci., 2014, 103 pp

18 The Contrarian Nature of Free Fatty Acid Particles Refrigerated vs. 25 C Storage Particle Morphology and Polysorbate Composition Silicone il is Helpful 18

19 Enhancing PS20 FFA particle formation 1% PS20 stock solutions with saturated free fatty acids M20: 98g water + 1g myristic acid + 1 g PS20 L20: 98g water + 1g lauric acid + 1 g PS20 Excess fatty acid removed by filtration. Test formulations: 140 mg/ml mabx, isotonic, ph << pi, 0.010% PS20 (control) 140 mg/ml mabx, isotonic, ph << pi, 0.010% L mg/ml mabx, isotonic, ph << pi, 0.010% M20 19

20 M20 L20 Refrigerated vs. 25 C Storage 30 days at 4 C ne additional day at 25 C 20

21 Prior data suggested a link between fatty acid type and FFA particle morphology Adapted from Siska, et al., J. Pharm. Sci., 2014 nline 21

22 The M20 and L20 data further supported this conclusion. (23 days, 4 C) M20 particles are consistently small, indeterminate shape L20 particles are larger, often rod-like 22

23 Protein is not a critical factor in FFA particle formation Placebo vial with M20 Placebo vial with L20 23

24 Lauric-acid particles consistently skew to larger sizes. FFA particles can be generated in the absence of protein and the morphology and size distribution matchobservations in protein-containing formulations. 24

25 Container dependence and the value of silicone oil The observation of FFA particles has been very consistent in glass vials. Glass pre-filled syringes have shown no evidence of FFA particle formation. Potential causes: Silicone oil is present in syringes, but not vials. Syringes present a more challenging target for inspection, making visualization of particles and/or analysis by MFI challenging. Initiated a study with M20 and L20 to evaluate the potential causes. 25

26 A study challenge near time zero. Syringes formulated with mabx and 0.01% M20. Syringes filled and placed immediately at 4 C Particles formed in 12 hours. (Syringe 1) Syringes filled, held at 25 C for 24 hours, then moved to 4 C. Temporary incubation prevented particulation. (Syringe 2) Note: visualization of FFA particles in Syringe 1 was not a challenge. 4 C 25 C 4 C

27 Syringes showed no FFA particles during 30-days of 4 C storage. L20 M20 L20 M20 27

28 Adding silicone oil to glass vials protected against FFA particles (30 days at 4 C) Standard Fill 55 µl Silicone il Float L20 M20 28

29 MFI results are consistent with visual observations Silicone oil addition to M20 and L20 vials reduces particle counts ( 1µm). Syringe particle counts are intermediate, potentially reflecting saturation of the limited amount of silicone oil present. M20 L20 29

30 What about non-glass syringes? Daikyo Crystal Zenith Syringe Cyclic polyolefin container with Fluorotec piston. Silicone oil free. Visual inspection challenging due to distortion. MFI particle counts show comparable particle counts to glass syringes. Barrel surface and/or piston may provide a sink for free fatty acids. M20 30

31 Do FFA particles actually matter? Meeting release/stability specifications. Difficult to track/quantify. Potentially a visual defect. Reversibility at 25 C creates potential variability. May reflect of loss of surfactant protection. Potentially driven by co-purified enzyme (Labrenz, J. Pharm. Sci. 2014). Chemical hydrolysis could affect long-term storage. May reflect changes in raw material quality. Initial levels of free fatty acids influence kinetics. Unknown effect from polysorbate heterogeneity. 31

32 Do FFA actually matter? Patient safety. The current industry focus on FFA particles reflects a robust interest in the relationship between particles and patient safety. The FFA particles appear to be largely solubility driven, dispersing at higher temperatures and with increased dilution (ie, prior to delivery and/or in vivo). To the extent that FFA particles interact with protein, they appear to drive large particles (Cao, et al., J. Pharm. Sci., 2014, online), trackable by standard analytical methods (ie, HIAC and visual inspection for visible particles). 32

33 Acknowledgements Christopher Pierini Lead for the silicone oil study. Support from Amgen Management Bruce Kerwin Michael Treuheit Ping Yeh The free fatty acid particle team and J. Pharm. Sci. article coauthors. Xiaolin Cao* Stephen Brych Gerd Kleemann Ramil Latypov Hollis Lau Jennifer Litowski* Christine Siska* Christopher Pierini* Zai-Qing Wen *primary contributors 33