Annick GERVAIS, UCB, Belgium on behalf of EBE

CASSS CMC Strategy Forum – EBE satellite session – Sorrento – May 5, 2014

Concept Paper on Forced

Degradation Studies

Background

2

• Early 2013 – EBE Biomanufacturing Working Group endorsed the project

of a Concept Paper for Forced Degradation Studies

• Aim of this Concept Paper: Describe approaches and best practices

amongst biopharmaceutical companies in the design and execution of

Forced Degradation Studies (FDS).

• May 2013 – Kick off meeting

• Working group composed of 9 EBE member Companies:

Janssen Biologics, MedImmune, Merck-Serono, Novo Nordisk, Pfizer,

Roche, Sanofi, UCB (topic group lead)

• Draft concept paper will be ready for review end Q3 2014

Objective & Scope of the Concept Paper

3

• Provide industry best practices in the field of Forced Degradation Studies

for therapeutic proteins based on experience of the EBE member

companies.

• Does not aim at defining regulatory requirements.

• The following questions will be addressed:

- Why do we need to use Forced Degradation Studies?

- When to initiate them?

- At which product development phase?

- Which are the relevant stressing conditions and for which purpose?

- What is the extent of degradation we want to achieve?

- How are acceptance criteria defined?

- What are the limitations of Forced Degradation Studies?

What is FDS and What is it Used For?

4

• FDS = intentional degradation of a molecule to an appropriate extent by means of

various stressing agents (temperature, light, chemical agents, mechanical stress).

The final purpose is to mimic what could happen under storage conditions and

to confirm that the stability indicating analytical methods are appropriate

• Note: in our definition of FDS, there is

an overlap with stressed stability conditions

when applying temperature as stressing agent

• FDS can provide

multiple information

Forced Degradation Studies

Possible Degradation

Pathways

Understand CQA Candidate

Selection

Manufacturing Process

Development

Formulation Development

Product Related Species

Characteri-sation

Comparability

Method Development

Stability Indicating Methods

Temperature Excursions

Storage / freeze-thaw

Goal of this Presentation and Presentation Outline

• To present specific case studies and related questions to initiate discussions

in the panel.

• The discussions will help the team finalising the draft Concept Paper.

Case Studies:

• Candidate Selection

• Comparability – How are acceptance criteria defined?

- Degradation pathway

- Kinetics of degradation

- Particular case of lyophilised products

• Photostability

5

CASE STUDY 1

Candidate Selection

6

When Are Forced Degradation Studies Performed?

sequence lock

No. clones 1011 100’s 10’s 1-2 109 100’s 10’s 1-2

FDS

Study

Example of tests performed:

- Tm,

- Aggregate levels on agitation,

- Temperature Study,

- Forced Oxidation and Deamidation,

- bioactivity

CASE STUDY 2

Comparability

How to define acceptance criteria for:

1. Degradation pathway « static » comparison

2. Kinetics of degradation comparison

8

Degradation Pathway – How to Compare Pre- and Post-change ?

• Monoclonal Antibody - Characterization for comparability between

pre and post change

- Time zero

- Post forced degradation by temperature (8 weeks at 40°C)

- 7 samples tested

3 pre-change + 1 reference STD

3 post change

Use of specific and orthogonal methods:

Qualitative and quantitative data

9

Degradation Pathway – Mab – Peptide mapping/MS – Qualitative information

Chain PTMs

Time zero Post Forced degradation

Ref.

STD

pre-

change

Batch 1

pre-

change

Batch 2

pre-

change

Batch 3

post

change

Batch 1

post

change

Batch 2

post

change

Batch 3

Ref.

STD

pre-

change

Batch 1

pre-

change

Batch 2

pre-

change

Batch 3

post

change

batch 1

post

change

batch 2

post

change

batch 3

HC Ox M (a) Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

HC Deam N(a) Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

HC Succ N(a)

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

HC Deam N(b) Yes

minor

Yes

minor

Yes

minor

Yes

minor

Yes

minor

Yes

minor

Yes

minor Yes Yes Yes Yes Yes Yes Yes

HC Deam Q(a) Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

minor

Yes

minor

Yes

minor

Yes

minor

Yes

minor

Yes

minor

Yes

minor

HC C-Term

Truncation Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

LC Deam N(c) No No No No No No No Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

HC Ox M(b) No Yes

Trace

Yes

Trace

Yes

Trace No No No

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

HC Ox M(c) No Yes

Trace

Yes

Trace

Yes

Trace No No No

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

Yes

Trace

HC Pyro-Glu (Q)

(N-TERM) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Acceptance criteria for

comparability : same PTMs and

similar intensity between pre

and post change before and

after forced degradation.

Acceptance criteria

MET

Same PTMs between

pre and post change

with similar intensities

10

Degradation Pathway – MAb – Deamidation – Quantitative Analysis

0 5

10 15 20 25 30 35 40 45

Ref. STD Pre change Batch 1

Pre change Batch 2

Pre change Batch 3

Post Change batch 1

Post Change batch 2

Post Change batch 3

% Deamidation Time Zero % Deamidation Post forced Degradation

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

Minutes

9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00

T=0

T= 8w

Deamidated species

Acceptance criteria MET

Acceptance criteria for comparability : same

deamidation ranges between pre and post

change before and after forced degradation.

11

• Behavior under thermal stress

- Better purity for PIII material

• Aggregation profile confirmed by orthogonal methods (AF4 & svAUC) A

U

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

0.034

0.036

0.038

0.040

0.042

0.044

0.046

0.048

0.050

0.052

0.054

0.056

0.058

0.060

0.062

0.064

0.066

0.068

0.070

Minutes

5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40

Post-change T0 & 4w 40°C

AU

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

0.034

0.036

0.038

0.040

0.042

0.044

0.046

0.048

0.050

0.052

0.054

0.056

0.058

0.060

0.062

0.064

0.066

0.068

0.070

Minutes

5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40

Pre-change T0 & 4w 40°C

Monom

er

dim

er

LM

W 1

LM

W 2

HM

W 1

HM

W4

HM

W 3

Degradation Pathway – Antibody Drug Conjugate – Size Variants

12

• Soluble aggregates

- Dimer increase

Slopes ratio (Post/Pre) = 100%

- Other HMWs

Slopes ratio (Post/Pre) = 96%

• Fragments

- Slopes ratio (Post/Pre) = 90%

Compliant with acceptance criteria:

80% to 125%

Acceptance criteria (proposed by FDA) :

comparison of slopes

slope ratio: 80% to 125%

Kinetics of Degradation – Antibody Drug Conjugate – Size Variants (1)

13

14

0 0.5 1 1.5 2 2.5 3

– to

3

–

Common slope/different

Intercept model

Degradation profiles behave

similarly between process 1

and process 2

Acceptance criteria : covariance analysis at p=0.25 (poolability criteria).

1. Test for the equality of slopes

2. Test for equality of intercepts

Kinetics of Degradation – Mab – Size Variants (2)

Kinetics of Degradation – Examples 1&2 – Acceptance Criteria – Limitations

• Both approaches require a linear model

• To be significant, the degradation must be sufficiently high in comparison

to the method variability.

15

Particular Case of Lyophilised Products - Pitfalls

• Lyophilised Drug Products are typically very stable

Practically no degradation is observed at recommended storage

condition

• In case substantial degradation is targeted, harsh stress conditions have to

be applied

• How representative are degradation products obtained at harsh stress

conditions?

• Is there a requirement to do FDS on lyophilised products?

or is it better to do it on the API?

• What can be done to show comparability on lyophilised products?

16

Conclusions (1)

Expectations from Health Authorities:

• Forced degradation studies are expected for comparison of pre- and post-

change stability profiles, especially for transitions from early to late clinical

development and post-approval comparability protocols.

• Meaningful forced degradation conditions should be selected which result

in controlled degradation.

• Conditions should be selected which allow sufficient degradation to

occur over a reasonable time period.

• Multiple time points are expected for each condition to determine a

slope (trend) accurately for each quality attribute measured.

• Example: Accelerated and stressed conditions for a lyophilized drug

product may not allow sufficient degradation to occur for comparison of

drug substance process changes.

17

Conclusions (2)

Industry Concerns:

• Extent of degradation to achieve in FDS should be close to degradation

seen over shelf-life to be meaningful

- But, statistical evaluation of limited degradation might be misleading

• To be significant, the degradation must be sufficiently high in comparison

to the method variability.

18

CASE STUDY 3

Photostability

19

Photostability – Evaluation by SEC analyses

• Exposure – ICH conditions

- 25ºC ≥ 1.2 million lux hours and ≥ 200

Watt hrs/m2: ICH Confirmatory conditions

- 25ºC ≥ 1.2 million lux hours: visible

conditions (sample protected by UV filter)

- 25°C Dark control: sample wrapped in

aluminum foil (time point taken at end of

each incubation)

- Reference Standard

• Exposure – Stressed Conditions

- 25ºC ≥ 2.4 million lux hours and ≥ 400

Watt hrs/m2: stress conditions.

- 25ºC ≥ 2.4 million lux hours: stress visible

conditions (sample protected by UV filter)

- Day 0 -10 in a photo-chamber

- Dark control

20

Rate of increase over time for aggregates 1,

2 & 3 and fragments

Photostability: ICH Conditions Versus a Time Course Incubation

21

Lane 3: DS01 Reference Standard

Lane 4: ≥ 1.2 million lux hour ± ≥ 200Wh/m2 (Vis + UV)

Lane 5: ≥ 1.2 million lux hour ± ≥ 200Wh/m2 (Vis only)

Lane 6: Dark control

22

Photostability – ICH Conditions – Cation Exchange Chromatography of

Samples Treated with Carboxypeptidase B

• Exposure – ICH conditions

- 25ºC ≥ 1.2 million lux hours and ≥ 200 Watt hrs/m2: ICH Confirmatory conditions

- 25ºC ≥ 1.2 million lux hours: visible conditions (sample protected by UV filter)

- 25°C Dark control: sample wrapped in aluminum foil (time point taken at end of each incubation)

- Reference Standard

Photostability - Conclusion

23

• ICH recommended conditions induce aggregation and fragmentation

• Time course photostability studies also induce aggregation and

fragmentation

• Easier to control the amount of aggregation and fragmentation using time

course studies, especially as the impact of photostress is molecule

dependent

• Impact of the stress on analytical techniques should be considered –

- Size based methods

- Consider the impact of photo-stress on surface charge and charge based

methods

- Others: mass spectrometry for post-translational modifications; Fluorescence

for unfolding etc

Acknowledgments

24

Concept Paper Working Group:

- Annick Gervais, UCB

- John O’Hara, UCB

- Karin Juul Jensen, Novo Nordisk

- Karin Sewerin, MedImmune (consultant)

- Mara Rossi, Merck-Serono

- Michael Nedved, Janssen Biologics

- Paul Dillon, Pfizer

- Shahid Uddin, MedImmune

- Stéphane Cornen, Sanofi

- Volker Schnaible, Roche

All reviewers and contributors from the EBE BioManufacturing Working

Group member companies experts.

Panel Members:

- Annick Gervais, UCB

- Carlo Giartosio, Merck-Serono

- Gabriella Angiuoni, Merck-Serono

- John O’Hara, UCB

- Shahid Uddin, MedImmune

- Stéphane Cornen, Sanofi

Chaired by:

- Karin Sewerin, MedImmune (consultant)

- Ronald Imhoff, Janssen Biologics

Open Questions for Panel Discussion

25

• Extent of degradation:

- What should be the « appropriate level of degradation »? What product characteristics are important to

assess?

- Extent of degradation should be close to degradation seen over the shelf-life to be meaningful, but to be

significant, the degradation must be sufficiently high in comparison to the method variability. How to deal with

cases where it is not?

- Traditionally, lyophilised products are considered very stable. How representative is the degradation profile for

lyophilised products?

• Comparability:

- Would it be helpful to conduct forced degradation at more than one temperature (e.g., 25C and 40C) to have

more controlled conditions across all of the analytical parameters?

- Besides using slope ratios, what other approaches could be used as a way to compare degradation rates?

- Apart from linear models, what other statistical model can be used?

- How many batches are required to demonstrate comparability? Are 3 batches sufficient ? What about

statistical evaluation? What about early development comparability where 3 batches may be difficult to have?

• Photostability:

- The standard ICH exposure time is very harsh. It might not fall under the category of “controlled degradation”

in some applications.

In general, would time course studies for photo-stress studies be more effective in determining the sensitivity to

fluorescent or UV light?

• Different oxidation mechanisms are induced by chemical, photo-stress, and metal-stress. Which

stresses are preferred for analytical method development to determine stability indicating

properties?

26

THANK YOU