Forced Degradation of Protein Therapeutics

Transcription

1 Forced Degradation of Protein Therapeutics Wim Jiskoot Division of Drug Delivery Technology Leiden Academic Centre for Drug Research (LACDR) AAPS Annual Meeting Orlando, FL 28 October 2015

2 Forced degradation (or stress testing): definition An umbrella term covering all forms of applying stress to drug substance or drug product exceeding the conditions used for stability testing For comparison: o o Stability testing: studies performed to assess the stability of a formulation according to the international requirements, in particular ICH guidelines Q1A (drugs in general) and Q5C (biotech products) Accelerated testing: stability testing at elevated temperatures under quiescent conditions, according to ICH Q1A and Q5C Hawe et al. (2012) J. Pharm. Sci. 101:

3 Forced degradation of therapeutic proteins: why? Candidate molecule selection Molecule characterization (pre-formulation) Formulation development Assay development and validation Shelf life setting Exposure to conditions other than intended Future product development Comparability studies (after formulation/process changes; biosimilar product development) Fundamental studies on degradation mechanisms Hawe et al. (2012) J. Pharm. Sci. 101:

4 Forced degradation: stress factors Elevated temperature Temperature fluctuations Freezing Freeze-thawing Mechanical stress (e.g., shaking, stirring, pumping) Light Oxidative stress ph changes Interfaces (e.g., air/liquid, liquid/container) X-ray Hawe et al. (2012) J. Pharm. Sci. 101:

5 Forced degradation: stress factors Elevated temperature Temperature fluctuations Freezing Freeze-thawing Mechanical stress (e.g., shaking, stirring, pumping) Light Oxidative stress ph changes Interfaces (e.g., air/liquid, liquid/container) X-ray Hawe et al. (2012) J. Pharm. Sci. 101:

6 Thermal stress Elevated temperature is the most widespread method to stress and degrade therapeutic proteins Variables: temperature (fluctuations), incubation time Stay beneath melting temperature o Protein dependent o Formulation dependent Determine thermal stability (T onset and T m ) with temperature ramp (DSC, DSF, etc.) NB not a forced degradation test! No standard conditions available Hawe et al. (2012) J. Pharm. Sci. 101:

7 Enbrel 50 mg/ml pre-filled syringe, 7 days 50 C Compared to day 0: ns = not significant, * p<0.05, ** p<0.01, *** p<0.001 Method day 1 day 2 day 3 day 4 day 7 Bis-ANS DLS, Zave DLS, PDI HP-SEC LO, >1 µm LO, >10 µm LO, >25 µm CD 222 nm UV 2 nd der *** *** *** *** *** ns ns *** *** *** ns ns *** *** * ns ** *** *** *** ns ns ns ns ns ns ns ns ns ns ns ns ** ns ns * ns *** *** *** ns ns ns ns *** ELISA ns ns ns * *** Van Maarschalkerweerd et al. (2011) Eur. J. Pharm. Biopharm. 78:

8 Bis-ANS and polysorbate intensity of emission maximum [a.u.] Bis-ANS fluorescence as function of polysorbate 20 concentration in 100 mm phosphate, ph 7.2, containing 1.0 mg/ml IgG HT+PS NS+PS20 placebo+ps polysorbate 20 concentration [%] Hawe et al. (2010) Pharm. Res. 27:

9 Bis-ANS fluorescence versus CCJV fluorescence Hawe et al. (2010) Pharm. Res. 27: Bis-ANS fluorescence is sensitive to polarity Molecular rotor CCVJ fluorescence is sensitive to viscosity

10 CCVJ and polysorbate intensity of emission maximum [a.u.] CCVJ fluorescence as function of polysorbate 20 concentration in 100 mm phosphate, ph 7.2, containing 1.0 mg/ml IgG HT+PS20 NS+PS20 placebo+ps polysorbate 20 concentration [%] Hawe et al. (2010) Pharm. Res. 27:

11 fluorescence intensity [a.u.] Hawe et al. (2010) Pharm. Res. 27: Use of CCVJ to analyze Humira 40 mg (contains 0.1% polysorbate 80) 5 mm CCVJ (Exc: 435 nm) min 65 C 10 min 60 C NS placebo emission wavelength [nm]

12 Freeze-thawing Waterville Valley, New Hampshire, 11 June 2015

13 Freeze-thawing Total storage time (%) Proportion of total storage time per temperature C 255 (87%) patients turned in their temperature logger) The proportion of the patients who stored their product for at least 2 hours consecutive time below 0 o C or above 25 o C was 24.3% (median: 3.7 hours) and 2.0% (median: 11.8 hours), respectively.

14 Freeze-thawing Sept 16 Sept 1 Oct 16 Oct March 16 March 1 April Vlieland et al. (2015), in press

15 Freeze-thawing: stress factors and conditions Stress factors Influencing conditions Interfacial stresses Freezing / thawing rates Temperature fluctuations Temperature Cryoconcentration Number of cycles Excipient crystallization Formulation Phase separation Volume ph shifts Container material Container geometry No standard conditions available Hawe et al. (2012) J. Pharm. Sci. 101:

16 Freeze-thawing: a case study 1.0 mg/ml monoclonal higg1 in 100 mm sodium phosphate, ph 7.2 Freeze-thaw stress: 5 cycles of -80 o C 25 o C Heating stress: 10 min 74 o C (a few degrees below T m )

17 Freeze-thawing: a case study Bis-ANS SDS-PAGE NS H FT NS H FT DCVJ Nile Red NS: nonstressed H: heated FT: freeze-thawed Extrinsic fluorescence

18 Freeze-thawing: a case study mean residual ellipticity [deg cm 2 dmol -1 ] Hawe et al. (2009) Eur. J. Pharm. Sci. 38: Far-UV circular dichroism C 5xFT NS wavelength [nm]

19 Freeze-thawing: a case study UV [mau] HP-SEC / A280 detection 5.0E E E E E-02 unstressed FT 10 min 75 C 5.9E E E E E E E time [min] Hawe et al. (2009) Eur. J. Pharm. Sci. 38: 79-87

20 Freeze-thawing: a case study Bis-ANS fluorescence [mau] HP-SEC / Bis-ANS fluorescence detection 1.2E E E-01 unstressed FT 10 min 75 C 5.9E E E E time [min] Hawe et al. (2009) Eur. J. Pharm. Sci. 38: 79-87

21 Freeze-thawing: a case study Hawe et al. (2009) Eur. J. Pharm. Sci. 38: Light obscuration Particles > 1 μm /ml Particles >10 μm /ml Nonstressed Heated 5x FT 0 Nonstressed Heated 5x FT

22 Freeze-thawing: a case study second derivative Second derivative FTIR spectrum of isolated aggregates xFT, prec. 77 C heat, prec. NS wavenumber [cm -1 ] Hawe et al. (2009) Eur. J. Pharm. Sci. 38: 79-87

23 Mechanical stress testing Stress methods Shaking Stirring Pumping Vortexing Sonication Special shearing devices Influencing conditions Rate and time Temperature Liquid-air interfaces Liquid-container interfaces Cavitation Container geometry Filling volume No standard conditions available Hawe et al. (2012) J. Pharm. Sci. 101:

24 Mechanical stress testing

25 Stir stress: a case study J. Pharm. Sci., in press

26 Stir stress: a case study 1 mg/ml monoclonal higg1 in PBS, ph 7.4 Magnetic stirring (300 rpm) Also, time-dependent increase in nanoparticle content Sediq et al. (2015) J. Pharm. Sci., in press

27 Stir stress: a case study Coomassie Blue staining after magnetic stirring: Protein adsorbs to container and stir bar Inhibited by adding polysorbate 20 Sediq et al. (2015) J. Pharm. Sci., in press

28 Stir stress: a case study 300 rpm 300 rpm balance Sediq et al. (2015) J. Pharm. Sci., in press

29 Stir stress: a case study Contact stirred IgG Non-contact stirred IgG, Contact stirred IgG + PS20, Non-stirred IgG, Contact stirred buffer Sediq et al. (2015) J. Pharm. Sci., in press

30 Stir stress: a case study Nanoparticle content Microparticle content Monomer content Sediq et al. (2015) J. Pharm. Sci., in press

31 Stir stress: a case study Intensity (AU) A) Fluorescent dye (DCVJ) fluorescence Enhanced fluorescence Contact stirred IgG Non-contact stirred IgG Unstirred IgG Heat stressed IgG Wavelength (nm) Sediq et al. (2015) J. Pharm. Sci., in press

32 Stir stress: a case study Fluorescent dye (DCVJ) fluorescence Sediq et al. (2015) J. Pharm. Sci., in press

33 Stir stress: a case study Proposed mechanism of contact stirring-induced aggregation Inhibited by surfactant Promoted by contact-stirring Sediq et al. (2015) J. Pharm. Sci., in press

34 Overall conclusions Forced degradation is applied for several reasons and plays a central role in the development of therapeutic proteins There are very few standard conditions ; experimental conditions should be chosen depending on the study aim Different stress factors lead to different degradation processes and products Extensive analytical characterization is an integral part of forced degradation studies Be cautious in extrapolating the outcome of forced degradation studies to real-life conditions Forced degradation studies cannot replace stability studies under real-time and real-temperature conditions

35 Acknowledgements Leiden University (Leiden, the Netherlands) Ahmad Sediq, Reza Nejadnik, Andreas van Maarschalkerweerd, Ruben Van Duijvenvoorden, Daniel Weinbuch Coriolis Pharma (Martinsried, Germany) Daniel Weinbuch, Andrea Hawe Michael Wiggenhorn F. Hoffmann La Roche (Basel, Switzerland) Hanns-Christian Mahler Joerg H.O. Garbe Sanquin Research (Amsterdam, the Netherlands) Gert-Jan Wolbink, Steven O. Stapel Univ. of Copenhagen (Copenhagen, Denmark) Marco van de Weert

36 Degradation during real-life conditions Thank you!