Generic approach to forced degradation in early phase to facilitate the development of

stability indicating methodsWCBP 2014, Washington DC, January 28-30 2014

Patrik Petersson and Jytte PedersenNovo Nordisk A/S, Måløv, Denmark

Presentation title Date 1

• Introduction• Protocol• Rationales• Examples

• Poster with experimental details

OutlinePresentation title Date 2

• Main purpose with the current work has been to generate samples for early phase development of stability indicating methods

• There are also other application areas …

IntroductionPresentation title Date 3

• Chemical degradation• Deamidation and hydrolysis at low/high pH• Light• Oxidation• Reduction

• Physical degradation (denaturation aggregation)• Freeze/thaw cycling• Mechanical stress• Chemical degradation• Temperature

IntroductionPresentation title Date 4

IntroductionPresentation title Date 5

LiteratureArticlesNN reports

People

ARTEk a lnln

Kinetic theory

ExperimentsIgG4 XInsulin X

Minimal no of experimentsMinimal sample consumption

“Generic” protocol for forced degradation

• Limited guidance from authorities and mainly for small molecules

• Several publications of protocols for small molecules• S. Klick et al. Pharm. Tech. Feb. 2005 (www.pharmtech.com)• K.M. Alsante et al, Separation Science and Technology, 3 (2001) 85-172

• For proteins no protocols but excellent reviews on degradation• A. Hawe et al. J. Pharm. Sci. 101 (2012) 895-913.• M. Hasija et al. Vaccine: Development and Therapy, 3 (2013) 11-33• W. Wang, Intl. J. Pharmaceutics 185 (1999) 129-188.

IntroductionPresentation title Date 6

Sample preparationPresentation title Date 7

• 1-5 mg/mL drug substance• 103 mM glycyl-glycine

Stocksolution

1.5 mL

Samples

• pH 3, 7, 9• pH 7 0.01 and 0.1% H2O2

• pH 7 0.1 and 1 mM dithiothreitol• pH 7 Light

3-87 µL

2M NaOH

2M HCl

5% H2O2

50 mM dithiothreitol

Reagents

Exposure to low/high pH and temp: ProtocolPresentation title Date 8

• Typically pH 3, 7 and 9 (depending on prior knowledge of solubility/stability)• 7 days at 5, 21 and 40°C

• Primary degradation pathways:• Deamidation – Asn (Gln)

• IEC, cIEF, (RPC for small proteins)• Hydrolysis - Asp, Asn, Ser

• RPC, SEC• Disulphide bond shuffling• Denaturation and aggregation

• SEC, SDS-PAGE, visual, turbidity

Insulin X

Ref.

pH 2 21°C

pH 2 40°C N/A severe aggregation

pH 7 40°C

pH 9 21°C

pH 9 40°C

0%

-4.7%

-3.1%

-2.6%

-25.6%

Rational: A “suitable” degree of degradation

• A suitable level according to literature• 10 to 20% for small molecules [1-4]• 15% for proteins [5]

Presentation title Date 9

-23%

-91%

_____________________[1] K.M. Alsante et al, Separation Science and Technology, Volume 3, (2001) 85-172.[2] R. Maheswaran, FDA perspective s: Scientific considerations of forced degradation studies in ANDA submissions, Pharm. Tech. , May 2, 2012 [3] S. Klick et al Pharm. Technol. 29 (2005) 48-66.[4] D.W. Reynolds et al, Pharm. Tech. Feb. 2002 (www.pharmtec.com)[5] A. Hawe et al. J. Pharm. Sci. 101 (2012) 895-913.

• A too extensive degradation willdegrade primary degradation products

• Insulin X and IgG4 X results suggests <20%

• We aim for 2-20%

Exposure to low/high pH and temp: RationalPresentation title Date 10

• Avoid denaturation and formation of insoluble aggregates• Work below the unfolding temperature (melting temp, tm)

85807676706160557163

Small proteins

Large proteins

IgG4s

tm at pH ~7 [°C]

____________[1] M. Hasija et al. Vaccine: Development and Therapy, 3 (2013) 11 – 33. [2] A. Hawe et al. J. Pharm. Sci. 101 (2012) 895-913.

• A rule of thumb tm – 10°C [1,2]

• tm from 55 to 85°C for 10 NN proteins

• <45°C should therefore be applicable

Exposure to low/high pH and temp: Rational

• Combined rate law and Arrhenius equations

• Rate law

• Arrhenius

Presentation title Date 11

0)/( lnln ctAec RTE a

RTE aAek

ktecc 0

-0.16

-0.14

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

0

0 5 10 15 20

25C experime

25C fitted

40C experime

40C fitted

time [days]

ln(c

/c0)

[-]

40°C

25°C

IgG4 X RPC pH 9

• Pseudo 1st order kinetics

• In agreement with literature [next slides]

Exposure to low/high pH and temp: Rational

• Models to predict a temp. and time resulting in a suitable degree of degradation

Presentation title Date 12

temperature, T time, t

20 40 6080 100 120

10 20 30 40 50 60

0

0.2

0.4

0.6

0.8

1

time [hours]temperature [C]

c/c0

[-] c/

c 0

c/c 0

Exposure to low/high pH and temp: RationalPresentation title Date 13

temperature (C ):time (days):

c/c0 %imp. c/c0 %imp. c/c0 %imp. 2-20%1 7.7E+04 1.8E+10 24 0.99 1 0.95 5 0.70 30 18 1.0E+05 1.7E+15 35 0.97 3 0.72 28 0.02 98 12 8.4E+04 1.2E+11 26 1.00 0 0.97 3 0.79 21 1

4.5 1.1E+05 8.6E+14 34 1.00 0 0.99 1 0.91 9 17 8.7E+04 1.2E+12 28 0.99 1 0.93 7 0.55 45 19 9.5E+04 2.4E+13 31 0.99 1 0.94 6 0.52 48 13 2.9E+04 3.5E+03 3 0.99 1 0.98 2 0.95 5 17 4.1E+04 9.9E+02 7 1.00 0 0.99 1 0.97 3 19 9.4E+04 1.3E+13 30 0.99 1 0.95 5 0.59 41 15 9.1E+04 6.5E+11 27 1.00 0 0.99 1 0.94 6 1

7.5 8.3E+04 3.9E+12 29 0.85 15 0.32 68 0.00 100 14 2.5E+05 7.4E+39 92 1.00 0 1.00 0 0.10 90 (1)

6.5 9.2E+04 3.5E+10 24 1.00 0 1.00 0 1.00 0 -9 8.4E+04 3.5E+10 24 1.00 0 0.99 1 0.94 6 14 2.8E+05 1.9E+45 104 1.00 0 0.99 1 0.00 100 (1)

6.5 4.6E+04 1.4E+04 10 1.00 0 0.99 1 0.96 4 19 8.9E+04 1.1E+12 28 1.00 0 0.97 3 0.79 21 12 0.55 45 (1)10 0.90 10 12 0.90 10 110 0.95 5 12 0.90 10 17 0.90 10 1

Protein X3 7.5 RPC 0.95 5 1

BSA 2 SECT.Estey et al J.Pharm.Sci. 95

(2006) 16260.625 38 (1)

57

217

407

J.C.M. Waterval et al, J. chromatogr. B, 754 (2001) 161.

A.Hawe et al, Pharmaceutical Research, 26 (2009) 1679

L.C. Gu et al, Pharmaceutical Research, 8 (1991) 485

K. Patel, Pharm. Res. 7 (1990) 703

IgG4 X

RPC

IEC

Peptide X1

Protein pH Ea [J/mol] A [1/h] ln(A) xx-LC Reference

Protein X2

RPC

RPC

RPC

Peptide X2

Protein X1

Interleukin

Oxytocin

Aplidine RPC

RPC

RPC

IEC• 10 proteins degraded at different pH = 25 combinations• 1 did not show any degradation• 20 resulted in 2-20% degradation (83%)• i.e. 7 days at 5, 21 and 40°C probably suitable• 4 resulted in >20% degradation new experiment at lower temp.

Exposure to low/high pH and temp: ProtocolPresentation title Date 14

• >20% degradation new experiment at lower temp.• Interpolation assuming pseudo 1st order kinetics

c/c0

0.7444

0.99230.9736

0

1

Insulin X

Exp. points at 5, 21 and 40°CAssume c/c0 = 1 at 1°C and 0 at 100°CJMP or Excel Solver function for non-linear fitting10 < ln(A) < 1005e4 < Ea < 5e5 J/molR 8.31 J/(mol K)t 168 h

• H2O2• Commonly used• Relevant for certain excipients e.g. polysorbate, PEGs

Presentation title Date 15

____________[1] Parr Instrument Company, Moline, Illinois, USA

Oxidation

• Azo compounds (R-N=N-R')• Alternative way to produce radicals

• O2• Most relevant but less commonly used• Oxygen bomb [1]• Cu2+, Fe3+, …

• Light• Relevant and commonly used

Light = O2 ≠ H2O2

Presentation title Date 16

• IgG4 X• RPC post reduction of S-S bonds

Reference = time 0

0.05% H2O2 21°C 2h

O2 30 bar 20°C 3 and 8 daysO2 40 bar 40°C 3 daysICHx1 and x3Reference = time 0

LCFc/2 Fd

Oxidation

O2 = Lightfor this protein

O2 = Light ≠ H2O2 for this protein

Oxidation by exposure to H2O2: ProtocolPresentation title Date 17

• Suggested exposure:• 0.01 and 0.1% H2O2 at pH 7, 21°C and 7 days

• Primary degradation pathways:• Oxidation

• Met, His, Trp, Tyr, Cys• RPC

• Aggregation• SEC, SDS-PAGE, visual, turbidity

Ref.

0.01% H2O2

0.1% H2O2

0%

-0.5%

-3.3%

0.95

0.96

0.97

0.98

0.99

1

1.01

0 0.05 0.1

c/c 0

[H2O2] [% w/v]Insulin X

Oxidation by exposure to H2O2: Rational

• Literature: 0.003-0.005% [1], 0.05% [2] and 0.01-0.1% [3]

Presentation title Date 18

____________[1] A. Hawe et al. J. Pharm. Sci. 101 (2012) 895-913.[2] J. Yu BipPharm, Nov. 2000 46–50. [3] M. Hasija et al. Vaccine: Development and Therapy, 3 (2013) 11 – 33.

[H2O2] [%] temp. [°C] time [h] c/c0 [-]Protein X1 0.003 20 25 0.40Protein X2 0.003 20 24 0.84Protein X3 0.025 20 24 0.85Peptide X1 0.09 25 504 0.90Protein X4 0.09 25 504 0.95Protein X5 0.1 21 168 0.96IgG4 X 0.1 20 2 0.85

0.7

0.75

0.8

0.85

0.9

0.95

1

0 0.1 0.2 0.3 0.4

% H2O2

c/c 0

IgG4 X

15% at 0.1% H2O2

00.10.20.30.40.50.60.70.80.9

1

0 50 100 150 200

Time [min]

c/c 0

2 h

• 6 of 7 NN proteins suggest that 0.01 or 0.1% is a suitable exposure• IgG 4 X results suggests a relatively fast reaction

Reduction by exposure to DTT: ProtocolPresentation title Date 19

• Suggested exposure:• 0.1 or 1 mM DTT at 21°C and pH 7 for 7 days• Dithiothreitol (DTT)• Non-denaturating conditions

• Primary degradation pathway:• Partial reduction of disulphide bonds

and thereby fragmentation or disulphide bond shuffling• RPC, SEC

0

0.2

0.4

0.6

0.8

1

0 0.5 1

c/c 0

[DTT] [mM]Insulin X

Ref.

0.1 mM DTT -23%

1 mM DTT -91%

Reduction by exposure to DTT: RationalPresentation title Date 20

• 3 of 4 NN proteins indicates that 0.1 or 1 mM DTT at 21°C pH 7 for 7 days gives a suitable degree of degradation

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20

Protein 2 (0.2 mM DTE)

Protein 3 (0.2 mM DTE)c/c 0

Time [days]

[DTT*] [mM] temp. [°C] time [h] c/c0Protein 1 DTE 0.2 25 168 0.96Protein 2 DTE 0.2 25 168 0.20Protein 3 DTE 0.2 25 168 0.98Protein 4 DTT 0.1 21 168 0.99Protein 4 DTT 1 21 168 0.97

* Dithiothreitol (DTT) or its epimer dithiothreitol (DTT)

Exposure to light: ProtocolPresentation title Date 21

• Suggested exposure: ICHx3 and x10 pH 7 and 25°C• ICHx3 = 600 watt hrs per square meter and 3.6 mill. lux hrs• ICHx10 = 2000 watt hrs per square meter and 12 mill. lux hrs• Suntester CPS+ [1]

• Primary degradation pathways: • Oxidation

• Met, His, Trp, Tyr, Cys• RPC

• Degradation of aromatic amino acids• Cys, Trp, Tyr, Phe• RPC

• Aggregation by covalentintermolecular cross links• SEC, visual, turbidity

____________[1] Atlas Material Testing Technology, Chicago, Illinois, USA

0.93

0.94

0.95

0.96

0.97

0.98

0.99

1

1.01

0 5 10

c/c 0

ICHxn

Ref.

Light ICHx3

Light ICHx10

0.1% H2O2

-3%

-6%

Insulin X

As for the IgG 4 light ≠ H2O2

Exposure to light: RationalPresentation title Date 22

• Literature - recommendations of >ICHx1 [1] or x1, x5 and x10 [2]

Vis. [Mluxh] 320-400 nm [Wh/m2]Protein X1 1.2 200 0.86Protein X2 1.2 200 0.98Protein X3 1.2 200 0.96Peptide X1 1.2 200 1.00Protein X4 1.2 200 0.98Protein X5 1.2 200 0.95Protein X6 1.2 200 1.00Protein X7 1.2 200 0.97Protein X8 12 2000 0.97IgG4 X 3.6 600 0.89

ICH light exposurec/c0

ICHx1

ICHx10ICHx3

__________________[1] M. Hasija et al. Vaccine: Development and Therapy, 3 (2013) 11-33[2] J. Yu BipPharm, Nov. 2000 46–50

• 7 of 8 NN proteins suggest that ICHx1 in most cases only results in a limiteddegradation, i.e. 0-5%

• Also and exposureto ICHx3 and x10 result in a limiteddegradation

Physical degradation

• Freeze/thaw cycling

• Mechanical stress• Shaking• Stirring

• Primary degradation pathways:• Aggregation after denaturation at hydrophobic interfaces

• Air/water interface• Ice/water interface• Surface of the container

• SEC, SDS-PAGE

• Sample handling problems and generation of relevant aggregates• More on physical degradation on the poster

Presentation title Date 23

Cost

• How much time?• Effective time ca 1 week• Total lead time ca 2 weeks

• How much drug substance is needed?• The entire protocol: 32 to 158 mg

• If access to drug substance is limited• Exclude conditions which are less relevant

• Physical degradation < DTT < H2O2 < light < low and high pH• Minimalistic approach = mid pH at 21 and 40°C (5 to 23 mg drug substance)

• Reduce volume to <1.5 mL at each condition

Presentation title Date 24

Conclusions

• Relatively general• Simple• Affordable• Forced degradation in early phase provides advantages

• Facilitates method development and validation• Provides knowledge about drug substance stability and degradation pathways• Gives input to mandatory late stage forced degradation studies• Provides input to process and formulation development• May facilitate characterisation of impurities• Meshes well with currently promoted QbD approaches

Presentation title Date 25