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Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 2
Examples of Bioproducts
Abbott Labs
GenentechAmgen/Pfizer
Eli Lilly
GenentechMerckGenentech
Eli Lilly
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 3
What is BioproductBioproducts are proteins produced from recombinant DNA and grown in an
expression system such as bacteria, yeast, or eukaryotic systems
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 4
• One of the key objectives in developing bioproduct is to find a commercial formulation prototype that has an acceptable stability profile throughout a desired shelf-life of 18 months or more, under typical storage condition of 2-8°C
• To expedite the decision process of selecting the optimal formulation prototype, a short-term accelerated stability is usually conducted by subjecting the formulation candidates to elevated multi temperature exposures (typically 15°C and higher)
• Based on this short-term multi temperature stability study, a prediction model is then developed to estimate the long-term stability profile of the formulation candidates under the intended long-term storage condition
Background
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 5
Why Stability Testing
• Safety point of view from patient• Critical quality attribute (CQA)• Establish shelf life of the drug• Study the storage conditions• Study the container closure system• Provide evidence how the quality of the drug product changes over
time
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 6
What’s so Special about Bioproduct Stability?Common problems with stability of proteins
• Usually sensitive to light, heat, air, and trace metal impurities• Small or large stress factors can disrupt protein folding• Numerous physical degradation routes, including agitation, freezing,
interaction with surfaces and phase boundaries• Possible Non-Arrhenius behavior• One type of degradation can facilitate other types of degradation
leading to a cascading effect• Possibility of different degradation mechanisms appearing depending
on the age of the product• Limited formulation options
Reference: Handbook of Stability Testing in Pharmaceutical Development. Anthony Mazzeo and Patrick Carpenter, Ch17, Stability Studies for Biologics
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 7
• Why accelerated stability studies work despite the problems listed on the previous slide?– Degradation is often reasonably Arrhenius below 40°C– Information from pre-formulation studies and other one-off studies
Bioproduct Stability
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 8
Challenges in Accelerated Stability Modeling
• When developing the prediction model from the short-term accelerated stability study:– Bioproducts typically degrade in a nonlinear fashion, numerous
chemical degradation routes possible, much more so than the small molecule compounds
– The underlying degradation mechanism is often very complex and a characterization study to understand the degradation kinetic is prohibitively expensive
– Limited resources to execute the accelerated study that minimal number of temperatures and testing time-points can be incorporated in the study design
• This presentation describes a proposal on how to develop the prediction model. Some key features:– Leveraging Arrhenius principle of the temperature dependence of a
chemical reaction
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 9
Accelerated Stability Study Design
• Key features of the stability design– Short-term, should be completed in 3 months or less– Typically utilize 4 temperatures at minimum: long-term storage
condition of 5°C + 3 elevated temperatures (15 – 40°C)– Highest temperature is chosen such that it is representative of
lower temperature stability profiles (e.g. elevated temperature degrades in the same pathway as the lower ones)
– Utilize materials (e.g. Drug Substance) that are representative of those for commercial use
– May incorporate other factors of interests besides temperature (e.g. pH, concentration of drug substance, choice of excipient, etc)
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 10
A Typical Accelerated Stability Study Sampling Scheme
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 11
Fixed Time-points vs. Fixed Amt of Change
• Fixed Schedule• Advantages:
• platform-wide approach (doesn’t need to vary with molecule)
• requires little prior knowledge• provides stability profile
• Disadvantages:• labor intensive• different levels of degradation at
different storage conditions – can bias rate coefficient estimates
• Fixed Degradant• Advantages:
• efficient• same level of degradation (rate coefficient
bias does not depend on storage condition)• Disadvantages:
• requires Ea estimate to design correct storage temperature / time-point combinations
• target degradant level must be selected a-priori
Months Months
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 12
Arrhenius Equation
• Rate constant k of a chemical reaction depends on the temperature (Kelvin) and activation energy Ea according to the following equation:
)ln(1
ln 0,Ra kTR
Ek
k = Reaction rate
Ea = Activation Energy (Kcal mol-1)
R = Gas constant (Kcal mol-1 K-1)
T = Temperature in Kelvin
kR,0 = Pre-exponential Factor
RTER
aekk 0,
ln (k)
1 / T
RE
Slope a
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 13
ln (k)
1 / T
RE
Slope a
Double Regression Analysis
• Step 1. Estimation of the k– Fit “Zero-order” regression of the concentration of an
analytical property vs. time, at each Temperature condition
• Step 2. Estimation of the Activation Energy– Fit Arrhenius Regression, using fitted k(T) values [i.e.,
slopes] from regression in Step 1 as Ys:
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 14
• High variability relative to degradation may lead to negative rate constant estimates that become truncated at the logarithm scale (e.g. increasing monomer ln(-x)=NA)
• Insufficient resolution (high degree of rounding) that results in the same value at each time-point can produce zero rate constant estimates (k=0) that become truncated (ln(0) -inf)
• Non-constant variance with the logarithmic form of Arrhenius
-6
-5
-4
-3
-2
-1
0
ln (
k)3.2 3.25 3.3 3.35 3.4 3.45 3.5 3.55 3.6
1000/T
Double Regression Analysis
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 15
Therapeutic proteins are complex molecules that can degrade (aggregate) via a variety of different physical/chemical mechanisms.
For simplicity, consider only two broad categories: reactivenon-reactive
time Only the ‘reactive’ monomer aggregates
Use first-order Arrhenius kinetics to describe the system
,0 exptotal NR R RM t M M k t
,0 ,0exp expR R a R ak k E RT k E RT
Parameters:Mtotal – total monomer concentrationt – timeMNR – non-reactive monomer concentrationMR,0 – reactive monomer concentration at t = 0kR – first-order rate constantkR,0 – Arrhenius pre-exponential termEa – apparent activation energykR,0’ = ln( kR,0 ) – for numerical convergence
A good compromise between first-principles rigor and practical limitations
Non-Linear Model Description
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 16
Non-Linear Model Fitting
time
Mto
tal
MNR
MNR + MR,0
Parameters:Mtotal – total monomer concentrationt – timeMNR – non-reactive monomer concentrationMR,0 – reactive monomer concentration at t = 0kR – first-order rate constantkR,0 – Arrhenius pre-exponential termEa – apparent activation energykR,0’ = ln( kR,0 ) – for numerical convergence
Model parameter is a function of…
Parameter
Analytical Property
Formulationa Temperature
MNR yes yes no
MR,0 yes no no
kR yes yes yes
kR,0, kR,0’ yes yes no
Ea yes no noa Formulation = unique set of pH, ionic strength, excipients; replicates of a given formulation (different “runs”) were fit independently.
The nonlinear regression platform in JMP 8.0.2 was used to estimate unknown
parameters
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 17
Double Regression vs. Non-Linear
91
92
93
94
95
96
97
98
99
Mo
no
mer
0 1 2 3
Month
5
25
30
40
TempC
90
92
94
96
98
100
Mo
no
mer
0 1 2 3
Month
Nonlinear Model
91
92
93
94
95
96
97
98
99
Mo
no
me r
0 1 2 3
Month
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
Ln
(K)
0 .0
03
2
0 .0
03
3
0 .0
03
4
0 .0
03
5
0 .0
03
6
1/T
Double Regression
Intercept
1/T
Term
25.849401
-7798.273
Estimate
Ea
K_r,0'
M_r,0
M_nr
Parameter
19.968332566
31.401137139
9.1844165116
89.793893802
Estimate
Ea=15.486
Company Confidential Copyright© 2013 Eli Lilly and Company
Slide 18
Concluding Remarks
• Summary– An approach for modeling the accelerated stability data for
biomolecules are presented– The nonlinear model based on the interplay between of reactive
and non-reactive species shown to fit the data quite well when there is sufficient degradation
• Future work– Evaluate alternative loss functions for better model selection (i.e.
goodness-of-fit)– Evaluate alternative nonlinear models– Find potential patterns from existing biomolecules that may provide
clues on how to better design and analyze data for future studies
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