Accelerated Stability During Formulation Development of Early Stage Protein Therapeutics

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


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


Reduced SDS-PAGE 6 months

blank10

30ºC, upright, 5 µg load9


5ºC, inverted, 5 µg load7




50 ng BSA Intensity Marker3

100 ngBSA Intensity Marker2

Mark 12 MWM1

SampleLane


blank10







50 ng BSA Intensity Marker3

Mark 12 MWM2

100 ngBSA Intensity Marker1

SampleLane

Non-reduced SDS-PAGE 6 months


>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 5


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



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



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


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


Formulation 3


15.00%17.00%19.00%21.00%23.00%25.00%27.00%

0 5 10 15

Time (months)

Peak

Are


Formulation 3


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

Accelerated Stability During Formulation Development of Early Stage Protein Therapeutics

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