forced degradation of protein therapeutics · pdf fileforced degradation (or stress testing):...

Division of Drug Delivery Technology Leiden Academic Centre for Drug Research (LACDR) AAPS

Annual Meeting Orlando, FL

28 October 2015

Forced Degradation of Protein Therapeutics

Wim Jiskoot

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 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)

o Accelerated testing: stability testing at elevated temperatures under quiescent conditions, according to ICH Q1A and Q5C

Hawe et al. (2012) J. Pharm. Sci. 101: 895-913

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


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: 895-913

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 (Tonset and Tm) with temperature

ramp (DSC, DSF, etc.) – NB not a forced degradation test!

No ‘standard conditions’ available


Enbrel® 50 mg/ml pre-filled syringe, 7 days 50°C

Method day 1 day 2 day 3 day 4 day 7

Bis-ANS *** *** *** *** ***

DLS, Zave ns ns *** *** ***

DLS, PDI ns ns *** *** *

HP-SEC ns ** *** *** ***

LO, >1 µm ns ns ns ns ns

LO, >10 µm ns ns ns ns ns

LO, >25 µm ns ns ** ns ns

CD 222 nm * ns *** *** ***

UV 2nd der ns ns ns ns ***

ELISA ns ns ns * ***

Compared to day 0: ns = not significant, * p<0.05, ** p<0.01, *** p<0.001

Van Maarschalkerweerd et al. (2011) Eur. J. Pharm. Biopharm. 78: 213-221

Bis-ANS and polysorbate

0.00 0.02 0.04 0.06 0.08 0.100

10000

20000

30000

40000

50000

placebo+PS20

NS+PS20

HT+PS20

polysorbate 20 concentration [%]

inte

nsit

y o

f em

issio

n m

axim

um

[a.u

.]

Bis-ANS fluorescence as function of polysorbate 20 concentration in 100 mM phosphate, pH 7.2, containing 1.0 mg/ml IgG

Hawe et al. (2010) Pharm. Res. 27: 314-326

Bis-ANS fluorescence versus CCJV fluorescence


Bis-ANS fluorescence is sensitive to

polarity

Molecular rotor CCVJ fluorescence

is sensitive to viscosity

CCVJ and polysorbate

0.00 0.02 0.04 0.06 0.08 0.100

2000

4000

6000

8000

10000

HT+PS20

placebo+PS20

NS+PS20

polysorbate 20 concentration [%]

inte

nsit

y o

f em

issio

n m

axim

um

[a.u

.]

CCVJ fluorescence as function of polysorbate 20 concentration in 100 mM phosphate, pH 7.2, containing 1.0 mg/ml IgG


Use of CCVJ to analyze Humira®40 mg (contains 0.1% polysorbate 80)

5 mM CCVJ (Exc: 435 nm)


450 500 550 6000

5000

10000

15000

20000

NS

10 min 60°C

placebo

10 min 65°C

emission wavelength [nm]

flu

ore

scen

ce i

nte

nsit

y [

a.u

.]

Freeze-thawing

Waterville Valley, New Hampshire, 11 June 2015

Freeze-thawing

0 2 ------------ 8°C

Proportion of total storage time per temperature 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 0oC or

above 25oC was 24.3% (median: 3.7

hours) and 2.0% (median: 11.8

hours), respectively.

Tota

l sto

rage

tim

e (

%)

Freeze-thawing

Vlieland et al. (2015), in press

0

-10

10

0

-10

10

1 March 16 March 1 April

1 Sept 16 Sept 1 Oct 16 Oct

Freeze-thawing: stress factors and conditions

Stress factors

• Interfacial stresses

• Temperature fluctuations

• Cryoconcentration

• Excipient crystallization

• Phase separation

• pH shifts

Influencing conditions

• Freezing / thawing rates

• Temperature

• Number of cycles

• Formulation

• Volume

• Container material

• Container geometry



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 -80oC – 25oC

• Heating stress: 10 min 74 oC (a few degrees below Tm)


SDS-PAGE

Extrinsic fluorescence

Bis-ANS

DCVJ

Nile Red

NS: nonstressed H: heated FT: freeze-thawed

NS H FT NS H FT


200 210 220 230 240-4000

-2000

0

2000

4000

NS

74°C

5xFT

wavelength [nm]

mean

resid

ual

ell

ipti

cit

y [

deg

cm

2 d

mol-1

]

Far-UV circular dichroism

Hawe et al. (2009) Eur. J. Pharm. Sci. 38: 79-87


-1.0E-02

1.9E-01

3.9E-01

5.9E-01

7.9E-01

9.9E-01

0 5 10 15 20 25 30

time [min]

UV

[m

AU

]

unstressed

FT

10 min 75°C

-1.0E-02

0.0E+00

1.0E-02

2.0E-02

3.0E-02

4.0E-02

5.0E-02

0 5 10 15 20 25 30

time [min]

UV

[m

AU

]

unstressed

FT

10 min 75°C

HP-SEC / A280 detection


-1.0E-02

1.9E-01

3.9E-01

5.9E-01

7.9E-01

9.9E-01

1.2E+00

0 5 10 15 20 25 30

time [min]

Bis

-AN

S f

luore

scen

ce [

mA

U]

unstressed

FT

10 min 75°C

HP-SEC / Bis-ANS fluorescence detection



Light obscuration

0

200

400

600

800

1000

1200

Nonstressed Heated 5x FT

Particles >10 μm /ml

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

Nonstressed Heated 5x FT

Particles > 1 μm /ml



1600 1620 1640 1660 1680 1700-0.3

-0.2

-0.1

-0.0

0.1

77°C heat, prec.

NS

25xFT, prec.

wavenumber [cm-1]

secon

d d

eri

vati

ve

Second derivative FTIR spectrum of isolated aggregates



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



Mechanical stress testing

Stir stress: a case study

J. Pharm. Sci., in press


Also, time-dependent increase in nanoparticle content

Magnetic stirring (300 rpm)

Sediq et al. (2015) J. Pharm. Sci., in press

1 mg/ml monoclonal hIgG1 in PBS, pH 7.4



Coomassie Blue staining after magnetic stirring: Protein adsorbs to container and stir bar Inhibited by adding polysorbate 20



balance

300 rpm 300 rpm


Contact stirred IgG

Non-contact stirred IgG, Contact stirred IgG + PS20, Non-stirred IgG, Contact stirred buffer



Nanoparticle content Microparticle content

Monomer content



450 500 550 600 650 7000

5.0106

1.0107

1.5107

2.0107

2.5107

3.0107

3.5107

Contact stirred IgG

Non-contact stirred IgG

Unstirred IgG

Heat stressed IgG

A)

Wavelength (nm)

Inte

nsit

y (

AU

)

Fluorescent dye (DCVJ) fluorescence


Enhanced fluorescence



Fluorescent dye (DCVJ) fluorescence


Proposed mechanism of contact stirring-induced aggregation


Promoted by contact-stirring

Inhibited by surfactant

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

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

Univ. of Copenhagen (Copenhagen, Denmark)

Marco van de Weert

Sanquin Research

(Amsterdam, the Netherlands)

Gert-Jan Wolbink, Steven O. Stapel

Thank you !

Degradation during “real-life” conditions

forced degradation of protein therapeutics · pdf fileforced degradation (or stress testing):...

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