2008 IBC Formulation Strategies for Protein Therapeutics, Accelerated Stability During Formulation Development of Early Stage Protein Therapeutics – Pros and Cons of Contrasting Approaches. Vice President, Biopharmaceutical Development Tim Kelly, Ph.D. KBI Biopharma, Inc.

1. Accelerated Stability During Formulation
Development of Early Stage Protein
Therapeutics – Pros and Cons of
Contrasting Approaches
2008 IBC Formulation Strategies for Protein Therapeutics
Tim Kelly, Ph.D.
Vice President, Biopharmaceutical Development
KBI Biopharma, Inc.

2. Overview
• Description of “Conservative” and “Aggressive”
approaches
• Tool Box Methodology
• Case Study: Aggressive Approach
• Case Study: Conservative Approach
• Pros and Cons of each Approach
• Conclusions

3. Stability Studies During Formulation
Development
• The goal of formulation development is to determine a
composition for the final dosage form that results in a
safe, efficacious product which remains stable over
the course of its intended use.
• Stability of various candidate formulations is generally
evaluated during development via a combination of
real-time and accelerated stability studies.
• Among drug companies, there is considerable
divergence in the extent of stability data deemed
appropriate to support formulation development during
preclinical and phase I clinical development.

4. Conservative Approach
• A conservative approach for evaluating formulation
stability may involve placing 10 or more candidate
formulations in a long term stability study under real-
time (e.g., 5°C) and accelerated (e.g., 30°C/65% RH)
conditions.
• May often include “platform” formulations for a given product
type
• The duration for such a study may range from 6
months to two years, with samples from each of the
candidate formulations analyzed in one to three month
intervals over the study duration.

5. Conservative Approach
• At the conclusion of the study, the candidate
formulation that exhibits the best overall conservation
of native purity and activity is chosen and utilized for
manufacturing of clinical trial material.
• This conservative approach is most commonly utilized
by large pharma companies with large product
development pipelines who have the time and
resources to permit this approach.

6. Conservative Approach
• Advantages
• Formulation decision is based on real-time stability data at
the intended product storage temperature.
» High degree of confidence that the product will remain stable
throughout the duration of its intended use in clinical studies.
» “Nothing substitutes for real-time stability”
• Disadvantages
• Time Consuming
• Labor Intensive
• Requires Large Quantity of API

7. Aggressive Approach
• Smaller pharma and biotech companies, especially
those who do not yet have revenue streams from
commercial products, often can’t afford to devote the
time and resources required to execute a 1-2 year real
time stability study during formulation development.
• Such companies may take a more aggressive
approach, with a greater emphasis on accelerated
stability over a shorter duration.
• KBI Biopharma frequently employs an approach
where a large number (30-40) of candidate
formulations are evaluated via statistical design of
experiments (DOE) including short term stability under
real-time and accelerated conditions for 0.5-2 months.

8. Aggressive Approach
• The accelerated stability conditions used in such
studies may range from 40-55°C to increase the rates
of degradation and enhance the likelihood of
observing significant differences among the candidate
formulations over the short duration of the study.
• Understand thermal properties (e,g., Tm’s of API) to set the
stress temperature
• A long term stability study may then be performed on
the selected formulation and ultimately on the actual
clinical trial material prepared in the final selected
formulation, in accordance with the ICH guidelines.

9. Aggressive Approach
• Advantages
• Acceleration of development timelines
• More efficient use of resources (people, lab, and API)
• Disadvantages
• Challenge of assigning predictive value to accelerated
stability data for protein therapeutics.
» Degradation processes that occur at accelerated temperatures
may be irrelevant to shelf life
• The translation of accelerated stability data into a real-time
storage shelf life is problematic as protein degradation
processes are quite complex and often do not follow
Arrhenius kinetics.
» Can not be used to directly predict real time shelf life

10. Tool Box Methodology
• Our study is only as good as our methods
• Biophysical, Analytical, Biological
• Interrogate thermal, conformational, chemical, and biological
stability
• Orthogonal Techniques
• e.g, SEC-HPLC and IEX-HPLC; DSC and DLS
• Conditions optimal for reducing formation of one impurity may be
sub-optimal for others
• Designed to detect/quantify product-related impurities of interest
• HMW species, deamidation, oxidation, other charge variants, clips /
truncated species
• Rugged with acceptable accuracy and precision, and understood
variability
• Differentiate analytical variability from changes to product
• Suitable for use in statistical DOE

11. Case Study – Aggressive Approach
• Monoclonal Antibody for IV administration
• ~25mg/mL
• Goal to initiate Phase I clinical trials as rapidly as
possible
• Goal to develop IV formulation within 6 months
• Preformulation Development
• Forced Degradation Studies
• Final Dosage Form Development
• Utilized aggressive approach to identify lead
candidate formulation based on statistical DOE, forced
degradation and accelerated stability

12. Case Study – Aggressive Approach
• Initial Linear Ranging Studies
• Solubility
• Biophysical characterization
• Forced Degradation Studies
• Freeze/Thaw
• Agitation
• Preformulation DOE
• Two 2-level factorial designs
• Two weeks accelerated stability
• Select Formulation
• 6 Month Non-GMP Stability Study on Selected
Formulation
• Stability on cGMP Drug Substance & Drug Product
Identify critical
factors,
Eliminate non-critical
factors

13. Case Study – Aggressive Approach
• Solubility
• Evaluated 5 buffer types, two pH values each
» Acetate, Citrate, Histidine, Succinate, Phosphate
• Selected 2 buffer types for further evaluation, based on
observed solubility and ease of concentration
• Forced Degradation
• Freeze-thaw and Agitation
• Evaluated excipients in each buffer system
» Salt, Sucrose, Sorbitol, Arginine, Histidine, Polysorbate 20
• Utilized Biophysical and Analytical techniques
» DSC, CD, FTIR, SEC-HPLC, CEX-HPLC, SDS-PAGE
• Selected 2 excipient types for further evaluation

14. Case Study – Aggressive Approach
• Accelerated Stability
• Two 2-level factorial designs
• 5°C (non-stressed) and 40°C/75%RH (stressed) for 2 weeks
• DSC, FTIR, SEC-HPLC, CEX-HPLC, SDS-PAGE, Bioassay
• Designed to optimize pH, buffer concentration, and
excipient concentration for each candidate buffer /
excipient system
• Evaluate interactions between formulation factors
• Interrogate thermal, conformational, chemical, and biological
stability using a combination of biophysical and analytical
techniques
• Understand and optimize the API design space
• DOE allows evaluation of statistical significance of
differences observed between candidate formulations

15. Use of DOE in Accelerated Stability
• DOE Summary
• Two factorial designs
• Selected to span a wide range of
pH conditions
• Full panel of analytics performed:
• SEC showed effect of buffer/pH
on HMW species
• Phosphate buffer: HMW
increases with increasing pH
• Histidine buffer: HMW stable from
pH 6 - 7
Design-Expert® Software
HMWspecies
Design Points
D1 Histidine
D2 Phosphate
X1 =A: pH
X2 =D: Buffer Type
Actual Factors
B: Buffer Conc =35.00
C: NaCl Conc =75.00
D: Buffer Type
6.00 6.25 6.50 6.75 7.00
Interaction
A: pH
HMWspecies
0.4
1.15
1.9
2.65
3.4
2
2

16. Case Study – Aggressive Approach
• Chose formulation and initiated a longer term (6
month) real time and accelerated stability study on the
selected formulation
• Performed concurrently with process development, scale up,
and Phase I bulk drug substance manufacturing
• Enabled and accelerated the transition to Phase I clinical
manufacturing
• Mitigated risk of short-term stability study via follow-on 6
month study
» Placed only the selected formulation up on stability for 6
months
» Extensive package of storage conditions
» -80°C, -20°C, 5°C Upright & Inverted, 30°C/60%RH Upright &
Inverted

17. Case Study – Aggressive Approach
SEC Main Peak
93.00%
94.00%
95.00%
96.00%
97.00%
98.00%
99.00%
0 2 4 6 8
Time (months)
PeakArea
-80C
-20C
5C upright
5C inverted
30C upright
30C inverted
SEC HMW Species
1.50%
1.60%
1.70%
1.80%
1.90%
2.00%
2.10%
2.20%
0 2 4 6 8
Time (months)
PeakArea(%)
-80C
-20C
5C upright
5C inverted
30C upright
30C inverted

18. Case Study – Aggressive Approach
CEX Acidic Species
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
0 2 4 6 8
Time (months)
PeakArea
-80C
-20C
5C upright
5C Inverted
30C upright
30C Inverted
CEX Main Peak
30.00%
35.00%
40.00%
45.00%
50.00%
55.00%
60.00%
65.00%
0 2 4 6 8
Time (months)
PeakArea
-80C
-20C
5C upright
5C Inverted
30C upright
30C Inverted
CEX Basic Species
4.00%
6.00%
8.00%
10.00%
12.00%
14.00%
0 2 4 6 8
Time (months)
PeakArea
-80C
-20C
5C upright
5C Inverted
30C upright
30C Inverted

19. Case Study – Aggressive Approach
Reduced SDS-PAGE 6 months
blank10
30ºC, upright, 5 µg load9
30ºC, upright, 20 µg load8
5ºC, inverted, 5 µg load7
5ºC, inverted, 20 µg load6
5ºC, upright, 5 µg load5
5ºC, upright, 20 µg load4
50 ng BSA Intensity Marker3
100 ngBSA Intensity Marker2
Mark 12 MWM1
SampleLane

20. Case Study – Aggressive Approach
blank10
30ºC, upright, 5 µg load9
30ºC, upright, 20 µg load8
5ºC, inverted, 5 µg load7
5ºC, inverted, 20 µg load6
5ºC, upright, 5 µg load5
5ºC, upright, 20 µg load4
50 ng BSA Intensity Marker3
Mark 12 MWM2
100 ngBSA Intensity Marker1
SampleLane
Non-reduced SDS-PAGE 6 months

21. Case Study – Aggressive Approach
>98% Main Peak
<2% HMW Species
>63% Main Peak
<28% Acidic Species
<10% Basic Species
cGMP Phase I Clinical Drug Product
6 Month Stability

22. Case Study – Conservative Approach
• Monoclonal Antibody for IV administration
• ~25mg/mL
• Goal to initiate Phase I clinical trials within 2-3 years
• Utilized a conservative approach with extensive real
time and accelerated stability
• Nine candidate formulations placed on stability for 6 months
• “Platform” formulation, primarily one buffer system; goal to
get some granularity within the buffer system (pH, excipients)
» Four temperature conditions between 5°C and 45°C
• Three top candidates selected and placed on stability for 12-
24 months
» Four temperature conditions between 5°C and 37°C
• SEC-HPLC, CEX-HPLC, SDS-PAGE, DLS

23. Case Study – Conservative Approach
SEC Main Peak, 5C
96.00%
96.50%
97.00%
97.50%
98.00%
0 2 4 6 8
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
Formulation 4
Formulation 5
Formulation 6
Formulation 7
Formulation 8
Formulation 9
SEC Main Peak, 25C
94.00%
94.50%
95.00%
95.50%
96.00%
96.50%
97.00%
0 2 4 6 8
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
Formulation 4
Formulation 5
Formulation 6
Formulation 7
Formulation 8
Formulation 9
SEC Main Peak, 37C
91.00%
92.00%
93.00%
94.00%
95.00%
96.00%
0 1 2 3 4
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
Formulation 4
Formulation 5
Formulation 6
Formulation 7
Formulation 8
Formulation 9

24. Case Study – Conservative Approach
CEX Main Peak, 5C
54.00%
54.50%
55.00%
55.50%
56.00%
56.50%
57.00%
57.50%
58.00%
0 2 4 6 8
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
Formulation 4
Formulation 5
Formulation 6
Formulation 7
Formulation 8
Formulation 9
CEX Main Peak, 25C
40.00%
45.00%
50.00%
55.00%
60.00%
65.00%
0 2 4 6 8
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
Formulation 4
Formulation 5
Formulation 6
Formulation 7
Formulation 8
Formulation 9
CEX Main Peak, 37C
25.00%
30.00%
35.00%
40.00%
45.00%
0 1 2 3 4
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
Formulation 4
Formulation 5
Formulation 6
Formulation 7
Formulation 8
Formulation 9

25. Case Study – Conservative Approach
CEX Acidic Species, 5C
20.00%
22.00%
24.00%
26.00%
28.00%
30.00%
0 2 4 6 8
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
Formulation 4
Formulation 5
Formulation 6
Formulation 7
Formulation 8
Formulation 9
CEX Acidic Species, 25C
20.00%
25.00%
30.00%
35.00%
40.00%
0 2 4 6 8
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
Formulation 4
Formulation 5
Formulation 6
Formulation 7
Formulation 8
Formulation 9
CEX Acidic Species, 37C
25.00%
30.00%
35.00%
40.00%
45.00%
50.00%
55.00%
60.00%
0 1 2 3 4
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
Formulation 4
Formulation 5
Formulation 6
Formulation 7
Formulation 8
Formulation 9

26. Case Study – Conservative Approach
Lane 1 – Mark 12 Molecular Weight
Standard
Lane 2 –Control
Lane 3 – 25ºC, Formulation 9
Lane 4 –5ºC, Formulation 9
Lane 5 –25ºC, Formulation 8
Lane 6 –5ºC, Formulation 8
Lane 7 – 25ºC, Formulation 7
Lane 8 –5ºC, Formulation 7
Lane 9 –25ºC, Formulation 6
Lane 10 –5ºC, Formulation 6
Non-reduced SDS-PAGE, 6 Months

27. Case Study – Conservative Approach
• Upon completion of 6 month study, three formulations selected and
placed on stability for 12 months
SEC HMW Species, 5C
0.50%
0.70%
0.90%
1.10%
1.30%
1.50%
1.70%
0 5 10 15
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
SEC HMW Species, 25C
0.50%
1.00%
1.50%
2.00%
0 5 10 15
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3

28. Case Study – Conservative Approach
CEX Main Species, 5C
40.00%
42.00%
44.00%
46.00%
48.00%
50.00%
52.00%
54.00%
0 5 10 15
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
CEX Main Species, 25C
40.00%
42.00%
44.00%
46.00%
48.00%
50.00%
52.00%
54.00%
0 5 10 15
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
CEX Acidic Species, 5C
15.00%
17.00%
19.00%
21.00%
23.00%
25.00%
27.00%
0 5 10 15
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3
CEX Acidic Species, 25C
20.00%
25.00%
30.00%
35.00%
40.00%
0 5 10 15
Time (months)
PeakArea
Formulation 1
Formulation 2
Formulation 3

29. Aggressive vs. Conservative
• Aggressive - Pros
• Delivered formulation composition in approximately 4 months
» Facilitated the completion of process development and scale-
up activities
» Completed 6 months of real-time non-GMP stability within 11
months of project initiation
» Plenty of real-time stability data to support IND
• Resulting formulation showed excellent 5°C stability
» 0.2% decrease in purity via SEC-HPLC after 6 months
» No significant changes to CEX-HPLC or SDS-PAGE profile
after 6 months
» Similar profile for cGMP drug product lot after 6 months storage

30. Aggressive vs. Conservative
• Aggressive - Cons
• No 5°C real-time stability data beyond 6 months
• Virtually no real-time stability data on “back-up” formulations
in the event of precipitous drop in purity after 6 months
• Statistical DOE and short term accelerated stability (2-
4 weeks) may be used to identify formulations with
acceptable long term real-time stability profiles
• Evaluate interactions between formulation factors to optimize
design space
• Use biophysical characterization techniques to assess
thermal and conformational stability of candidate
formulations

31. Aggressive vs. Conservative
• Conservative - Pros
• Resulting formulations showed excellent 5°C stability
» ≤0.5% increase in HMW species via SEC-HPLC after 12
months for all three formulations
» ≤4% decrease in MP purity via CEX-HPLC after 12 months for
all three formulations
• High level of confidence in ultimate shelf life of clinical
supplies
• Strong “fall back” position of alternative formulations with
extensive real-time stability to support their use should
problems arise during manufacturing or early clinical
development
• Plenty of real-time stability to support IND

32. Aggressive vs. Conservative
• Conservative – Cons
• Observed very similar profiles for most formulations
» ≤0.5% difference in HMW species via SEC-HPLC after 12
months for all three formulations
» ≤4% difference in MP purity via CEX-HPLC after 12 months for
all three formulations
• Value relative to resources expended and time spent
• Little/no evaluation of different buffer/pH systems
» Platform approach, did not screen a broad range of buffers / pH
values

33. Aggressive Approach

34. Conservative Approach

35. Conclusions
• MAb formulations with acceptable long term stability profiles may
be developed via both aggressive and conservative approaches
• While the aggressive approach includes some elements of risk, it
offers substantial potential benefits with respect to timelines and
resources
• Statistical DOE and short term accelerated stability (2-4 weeks)
may be used to identify formulations with acceptable long term
real-time stability profiles
• The conservative approach offers significant risk mitigation via
“back up” formulations
• A comprehensive toolbox of orthogonal analytical and biophysical
techniques is essential in both cases, but particularly for the
aggressive approach

36. Acknowledgements
• Vickie Dowling, PhD, Group Leader,
Biopharmaceutical Development
• Khurshid Iqbal, PhD, Senior VP & CSO

37. Use of DOE in Accelerated Stability
• Linear response surface design
• Polydispersity index (PDI) via DLS
• Interaction between pH and excipient type
Design-Expert® Software
PDI
Design Points
C1 200mM Sorbitol
C2 125mM NaCl
X1 = A: pH
X2 = C: Excipient
Actual Factor
B: Buffer = Acetate
C: Excipient
4.25 4.88 5.50 6.13 6.75
Interaction
A: pH
PDI
-0.160017
0.0224876
0.204992
0.387496
0.57

38. Case Study – Aggressive Approach

39. Case Study – Conservative Approach

40. Case Study – Conservative Approach

41. Case Study – Conservative Approach

42. Case Study – Conservative Approach