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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.
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
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.
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.
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.
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
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.
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.
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
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
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
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
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
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
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
HMW species
Design Points
D1 HistidineD2 Phosphate
X1 = A: pHX2 = D: Buffer Type
Actual FactorsB: Buffer Conc = 35.00C: NaCl Conc = 75.00
D: Buffer Type
6.00 6.25 6.50 6.75 7.00
Interaction
A: pH
HM
W s
peci
es
0.4
1.15
1.9
2.65
3.4
2
2
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
Case Study – Aggressive ApproachSEC Main Peak
93.00%94.00%95.00%96.00%97.00%98.00%99.00%
0 2 4 6 8
Time (months)
Peak
Are
a -80C
-20C
5C upright
5C inverted
30C upright30C 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)
Peak
Are
a (%
)
-80C
-20C
5C upright
5C inverted
30C upright
30C inverted
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)
Peak
Are
a -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)
Pea
k A
rea -80C
-20C5C upright5C Inverted30C upright30C Inverted
CEX Basic Species
4.00%
6.00%
8.00%
10.00%
12.00%
14.00%
0 2 4 6 8
Time (months)
Peak
Are
a
-80C-20C5C upright5C Inverted30C upright30C Inverted
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
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
Case Study – Aggressive Approach
>98% Main Peak<2% HMW Species
>63% Main Peak<28% Acidic Species<10% Basic Species
cGMP Phase I Clinical Drug Product6 Month Stability
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
Case Study – Conservative ApproachSEC Main Peak, 5C
96.00%
96.50%
97.00%
97.50%
98.00%
0 2 4 6 8
Time (months)
Peak
Are
a
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)
Peak
Are
a
Formulation 1
Formulation 2Formulation 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)
Peak
Are
a
Formulation 1
Formulation 2
Formulation 3
Formulation 4
Formulation 5
Formulation 6
Formulation 7
Formulation 8
Formulation 9
Case Study – Conservative ApproachCEX 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)Pe
ak A
rea
Formulation 1
Formulation 2
Formulation 3Formulation 4
Formulation 5
Formulation 6Formulation 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)
Peak
Are
a
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)
Pea
k A
rea
Formulation 1Formulation 2Formulation 3Formulation 4Formulation 5Formulation 6Formulation 7Formulation 8Formulation 9
Case Study – Conservative ApproachCEX Acidic Species, 5C
20.00%
22.00%
24.00%
26.00%
28.00%
30.00%
0 2 4 6 8
Time (months)
Peak
Are
a
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)
Peak
Are
a
Formulation 1Formulation 2Formulation 3Formulation 4Formulation 5Formulation 6Formulation 7Formulation 8Formulation 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)
Pea
k A
rea
Formulation 1Formulation 2Formulation 3Formulation 4Formulation 5Formulation 6Formulation 7Formulation 8Formulation 9
Case Study – Conservative Approach
Lane 1 – Mark 12 Molecular Weight StandardLane 2 –ControlLane 3 – 25ºC, Formulation 9Lane 4 –5ºC, Formulation 9Lane 5 –25ºC, Formulation 8Lane 6 –5ºC, Formulation 8 Lane 7 – 25ºC, Formulation 7Lane 8 –5ºC, Formulation 7Lane 9 –25ºC, Formulation 6Lane 10 –5ºC, Formulation 6
Non-reduced SDS-PAGE, 6 Months
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)
Peak
Are
a Formulation 1
Formulation 2
Formulation 3
SEC HMW Species, 25C
0.50%
1.00%
1.50%
2.00%
0 5 10 15
Time (months)
Pea
k Ar
ea Formulation 1Formulation 2Formulation 3
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)
Peak
Are
a Formulation 1Formulation 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)
Peak
Are
a Formulation 1Formulation 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)
Peak
Are
a Formulation 1Formulation 2
Formulation 3
CEX Acidic Species, 25C
20.00%
25.00%
30.00%
35.00%
40.00%
0 5 10 15
Time (months)
Peak
Are
a Formulation 1
Formulation 2
Formulation 3
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
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
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
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
Aggressive Approach
Conservative Approach
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 biophysicaltechniques is essential in both cases, but particularly for the aggressive approach
Acknowledgements
• Vickie Dowling, PhD, Group Leader, Biopharmaceutical Development
• Khurshid Iqbal, PhD, Senior VP & CSO
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
Des ign Points
C1 200m M SorbitolC2 125m M NaCl
X1 = A: pHX2 = C: Excipient
Actual FactorB: 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
Case Study – Aggressive Approach
Case Study – Conservative Approach
Case Study – Conservative Approach
Case Study – Conservative Approach
Case Study – Conservative Approach