Accelerated Stability Assessment:

Stability Studies of in vitro Diagnostic

(IVD) Products

Xiaodong (Jerry) Zhao

Beckman Coulter

Carlsbad, California

Content of this workshop

The importance of the evaluation of product stability

The objectives of product stability studies

The types of common stability studies

The designs of product stability tests

Kinetics parameters in stability tests

Accelerated product stability studies

Principle of accelerated stability studies

Applications of accelerated stability studies

Arrhenius equation: temperature and reaction rate

The empirical Q10 rule

Case studies

Medical products oversight by FDA

IND

NDAIND

BLA

510k

PMA

IDE

FDA

NCTRNational Center for

Toxicological Research

CVMCenter for Veterinary

Medicine

CDERCenter for Drugs

and Evaluation Research

CBERCenter for Biologics and

Evaluation Research

CDRHCenter for Devices and

Radiological Health

CFSANCenter for Food Safety

And Applied Nutrition

Devices

IVD

Drugs Biologics

Food

Drugs

Medical devices*

Biologics

Animal feed and drugs

Cosmetics

Radiation-emitting products*

Combination products

Drug-Device*, Biologic-Device*, Drug-Biologic

Office of Regulatory Affairs (ORA) is the office for all field activities

*

FDA recalls due to noncompliant stability issues

Stability data does not support expiration claims

The incorrect testing method was used

The incorrect expiration date was on the label

Stability data did not support expiration dating

Consequences of non-compliance

Issuance of warning letter

Endure a consent decree

Loss of certification to market the product

Stability characteristics: critical quality

attributes (CQA) of pharmaceutical/diagnostic

products

Assurance of the safety, efficacy and performance of a product during its shelf life

Legal requirement by regulatory agencies (ICH, FDA, WHO) and meeting

cGMP/QSR requirement

Safeguard the reputation of the manufacturer by providing the data to support the

claims of the marketed product

Objectives of stability studies

Establishing storage conditions that define how a product changes over time

under critical environmental factors

Revelation of the chemical nature of the decomposition and the mechanism of

the chemical transformation

Determining product specifications

Selecting container closure system for the product package

Justifying expiry dating of on market product

If the product stability changes beyond the claimed acceptance criteria,

established safety and efficacy claims are no longer applicable

Types of stability studies of IVD products

Shelf-life stability studies

Transport simulation studies

Winter stress cycle

Summer stress cycle

Shock, vibration, drop test

Temperature excursion stability studies (storage)

Freeze-thaw cycle

Extreme high temperature

Anti-microbial stability test

preservative efficacy and microbial count and challenges

Functional performance test

Closed- and open-container (in-use) stability

Claimed shelf-life

At least three lots manufactured under routine production conditions

The study report includes the protocol, lot numbers, acceptance criteria and testing intervals

The accelerated studies are accompanied by real time studies

The conclusion of the study validates the claimed shelf life

In-Use stability

Three lots tested under actual routine use of device (real or simulated). This may include assessing open vial stability, automated instruments, and on board stability.

The study report includes the protocol, lot numbers, acceptance criteria and testing intervals

The conclusion of the study validates the claimed in use stability

Design of stability studies

Full testing design:

Three batches of each strength, each package configuration for all time points

Test all specification parameters

Bracketing design:

Test at the extreme border conditions to support the configurations in between to

reduce the number of stability tests

Matrixing design:

Cover three batches of all conditions

But test a subset of a total number of possible samples for all combinations to

decrease the number of time points of tests

The types of stability parameters to be

considered during shelf-life

Chemical degradation

Each active ingredient stays within the specified limit (>90%)

Physical

Appearance: precipitation, content uniformity, clarity, moisture contents, particle size, package integrity, etc.

Microbiological

Sterility, resistance to microbial growth

Toxicological

No increase of toxicity

Performance

Unchanged therapeutic and diagnostic activity

The chemical stability of the products:

The factors effecting chemical stability: temperature, moisture, light, oxygen, pH

and package configuration

The chemical transformations during storage: oxidation, hydrolysis, isomerization

5-Chloro-2-methyl-4-

isothiazolin-3-one (CMIT)

2-Methyl-4-isothiazolin

-3-one (MIT)

Proclin 300Dowicil 200 Cosmocil

Polyhexamethylene biguanide (PHMB)1-(3-Chloroallyl)-3,5,7-triaza-

1-azoniaadamantane chloride

S-(+)-Ibuprofen

(S)-(+)-2-(4-Isobutylphenyl)propionic acid

Kinetics of chemical decomposition

The study of the rate of change and the way in which the rate is influenced by the

concentration of reactants, and other factors such as solvents, catalyst, pressure

and temperature

It can be measured by determining the change in the concentration of reactants

or products as a function of time

It allows a prediction of the degree of change at a given time

It gives insight into the mechanism of the reaction

The common degradation reaction in stability studies follows zero- or first-order

kinetics

Zero-order reaction

When the rate of reaction is independent of concentration of reactants. The

reaction follows zero-order kinetics

The rate of the zero-order kinetics is described as

[A]0

Time (t)

[A]

[A]=[A]0-kt

-k = slope90%

50%

A[A] B[B]k

Rate = -d[A]/dt = k [A] 0 = k

-d[A] = k dt

-d[A] = k dt

[A]0-[A]t = kt

t = ([A]0-[A]t)/k

[A]0 = initial concentration

[A]t = concentration at time t

t

0

Calculation of half-life and shelf-life of zero-

order reaction

[A]0

Time

[A]

[A]=[A]0-kt

-k = slope90%

50%

Determination of t1/2: [A]t = [A] 0/2

Substitute in the equation: t = ([A]0-[A]t)/k

t1/2 = ([A]0-[A]t)/k = ([A]0-0.5[A]0) /k = 0.5[A]0/k

Determination of shelf life t90% : [A]t = 0.9[A]0

t90% = ([A]0-[A]t)/k = ([A]0-0.9[A]0) /k = 0.1[A]0/k

k = concentration/time

= (mole/liter) day-1

First-order reaction

When the reaction rate is directly proportional to the first power of the

concentration of a single reactant, the reaction follows first-order kinetics

The rate of the first order reaction is described as

A[A] B[B]k

Rate = -d[A]/dt = k [A]

-d[A]/[A] = k dt

-d[A]/[A] = k t

ln[A]0 - ln[A]t = kt

t = ln([A]0/[A]t)/k

[A]0 = initial concentration

[A]t = concentration at time t

t

0

tln[A]0

Time

ln[A]

ln[A] = ln[A]0-kt

-k = slope

90%

50%

[A]0

[A]

90%

50%

Time

1) Determination of t1/2: [A]t = 0.5[A]0

Substitute in the equation: t = ln([A]0/[A]t)/k

t1/2 = ln([A]0/0.5[A]0)/k = ln2/k = 0.693/k

2) Determination of shelf life t90% : [A]t = 0.9[A]0

t90% = ln([A]0-0.9[A]0) /k = ln1.11/k = 0.105/k

k = time-1 = day-1

[A]0

t

[A]

90%

50%

ln[A]0

time

ln[A]

ln[A] = ln[A]0-kt

-k = slope

90%

50%

Calculation of half-life and shelf-life of first-

order reaction

Real time stability study vs accelerated

stability assessment program (ASAP)

Real time stability conducted at the claimed storage condition:

Store under selected conditions for longer than the expected shelf life (days, weeks, months)

Direct measurement of the real course of the change of the products

Check at regular intervals to see the trend

Benefit: direct conclusion, no calculation

Accelerated stability assessment program (ASAP):

Store the product at the elevated storage temperature, or other stressful environment

The rate of decomposition is increased and the trial period is shortened

Benefit:

Approximate ¼ of expected shelf life

See the impact of any changes much sooner

Application of real-time stability monitoring

A regulatory requirement as part of product license/clearance

As part of a manufacturer’s routine product quality assurance practice

To verify claims following changes to product formulation or manufacturing

As part of a corrective and preventive action (CAPA) plan for the product

(effectivity check)

Application of accelerated stability testing

Assessing the impact of multiple changes to a product formulation; Comparing the

relative effectiveness of different product formulations or container closure systems

This provides an early indication of the product shelf life and shortening of the

development schedule.

Testing at relatively high temperatures and/or humidity in early stages to determine

the type of degradation products which may be found after long-term storage

Establishing knowledge of stability failure modes for design risk analysis

Providing initial estimates of product stability through use of the Arrhenius equation

or other suitable data analysis that is predictive for the product under study.

The results obtained from accelerated testing can be used only as supportive stability

data.

Arrhenius equation: the relationship between the

rate constant and the temperature

lnk1

k2 =T1

1

T2

1

R

Ea

ln k = ln A -Ea

RTk =

-Ea

A·e RT

Arrhenius equation

These equations describe the relationships between the storage temperatures

and the degradation rates.

When the activation energy is known, the degradation rate at low temperature

may be projected from those observed at “stress” temperature.

Accelerated stability testing

A product is stressed at several higher temperatures. The information of the

product decomposition is projected to predict shelf life at the recommended

storage temperature.

For statistical reasons, the treatment in accelerated stability projections is

recommended to be conducted at four different stress temperatures.

The duration of the studies should be adequate to observe significant product

degradation, typically, two to six weeks, depending on the temperatures

selected.

Estimation of shelf-life and expiration date

Stability data collection: six time points in the study.

Data analysis: regression line based on calculation of 95% one-side confidence limit.

Expiry date: Border concentration is considered as the lowest specification limit and the point where the extension line cuts the 95% confidence limit is taken as an expiry date. Beyond this point, the product may no longer retain fitness for use.

Shelf life is the time during which the product will retain fitness for use (>90% of label claim of potency) if stored appropriately as per the manufacturer’s instruction.

Upper 95% CI Line

Lower 95% CI Line

Shelf life: 65 days

Day

Acceptance criteria:>5 mg/dl

An

alyt

e(m

g/d

l)

Iris iChemVELOCITY

Automated urinalysis chemistry solution

iRICELL®—Comprehensive urine testing

solutions

Iris iQ 200

Particle counts in urine by iQ 200

Strip chemistry in urine by iChemVELOCITY

Acceptance criteria of urine chemistry controls:

example of IRISpecCA control

Data analysis of the analytes in the IRISpecCA

control

The Rule of Q10: a useful tool for ASAP

A practical method: Q10 rule

The degradation reaction has to follow zero- or first-order kinetics

The principle of the Q10 rule is based upon the Arrhenius equation

The same model is used to fit each temperature

The duration should be adequate to observe significant product degradation,

typically, two to six weeks, depending on the temperatures selected

The Q10 Rule

Q10 is a number without a unit

Q10 is the factor by which the rate increases when the temperature is raised by

10 degrees

Conventional assumption for typical chemical reaction: Q10 values are ~2

Q10 =k2

k1

10T2-T1

A case study: activation energy of the

chemical transformation with a specified Q10

Ea = =

×T1T2

T2-T1

=k1

k2R×T1×T2×ln

T2-T1

R×lnk1

k2R×ln

1 1T1 T2

k1

k2

=8.314J/molK×298K×308K×ln2

10K= 52.9 kJ/mol

Q10 1.7 2 3

Ea (kJ/mol) 40.7 52.9 84.2

The Q10 Rule: a shortcut method for predicting

shelf-life of the products

Q10 Rule: a common practice of manufacturers for complex processes. But this

is not the official practice of ICH or FDA.

The Q10 rule states that a product degradation rate decreases by a constant

factor Q10 when the storage temperature decreases by 10C. The value of Q10

is typically set at 2 or 3 because these correspond to reasonable activation

energies. This model falsely assumes that the value of Q does not vary with

temperature.

The activation energy assumes the range between 10 and 20 kcal (41.8 kJ to

83.6 kJ) for a chemical decomposition. The use of 10 to 20 kcal is reasonable

because broad experience indicates that most analytes and reagents of interest

in pharmaceutical and clinical laboratories have activation energies in this

range.

A case study: estimation of the shelf-life of iQ

Calibrator with ASAP

T1 T2 T3

Temperature (C) 5 15 25

k (h-1) 1.6x 10-5 3.6x10-5 7.9x10-5

Component Test Specifications

Control KitControl run on

iQ200Pass

iQ Calibrator Coulter Count1200 ± 100

cells/μL

How Q10 value is used in an example

Q10 value is calculated as 2.2 not 2

We had a study run at 25C. We found

the product that tested at 25C expired

after 7 weeks. Based on Q10 = 2.2, we

would project the shelf life at refrigerated

conditions (5C) of 7x2.2x2.2 = 34 weeks

(8.5 month).

Shelf Life of iQ Calibrator

Time Point and Run Date

QC Release

24-Jul-15

Month 2

23-Sept-15

Month 4

17-Nov-15

Month 6

13-Jan-16

Month 8

15-Mar-16

Month 9

08-Apr-16

Days from Manufacture: 11 72 127 184 246 270

Daily Control Run

(Must Pass)Pass Pass Pass Pass Pass Pass

iQ Calibrator Coulter Count

(1200 ± 100 cells/μL)1196 1153 1142 1160 1108 1104

Q10 =k2

k1

10T2-T1

=0.0000360.000016

1015-5

= 2.25

Q10 =k3

k2

10T3-T2

=0.000079

0.000036

1025-15

= 2.19

A case study: Bilirubin failure happens prior to

the end of expiry of CA control

Bilirubin is the leading cause of the stability issues of CA control

Bilirubin is an analyte in urine from the

catabolism of red blood cells

Heme Biliverdin Bilirubin

Biliverdin

ReductaseHeme

Oxygenase

[O]

Bilirubin is sensitive to oxygen and light

Bilirubin is sensitive to oxidation during the reagent manufacturing and storage.

Bilirubin can be oxidized in the presence of dissolved oxygen (DO)

Design change in the filling process

Oxygen removal

Sparging Argon into the bottle while filling the reagent

Filling more reagent to limit the head-space in the bottle

106mL/123mL 120mL/123mL 123mL/123mL

Parameters used for evaluation of the stability

Bilirubin

Reflectance on the device

UV-Vis Absorption on the spectrophotometer

Dissolved Oxygen (DO)

DO probe

Bilirubin

Biliverdin

HPLC trace

The reported value of Bilirubin is correlated

with both analyte and degradation product

y = -48.499x + 62.174R² = 0.951

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

0.0000 0.1000 0.2000 0.3000 0.4000 0.5000 0.6000 0.7000 0.8000

Refl

ecta

nce f

rom

iQ

Velo

cit

y

Bilirubin (459 nm)

#7 DN LFR 45CMean BIL %R vs. Bilirubin A 459 nm

y = 114.89x + 13.273R² = 0.9626

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000

Refl

ecta

nce f

rom

iQ

Velo

cit

y

Biliverdin (365 nm)

#7 DN LFR 45CMean BIL %R vs. Biliverdin A 365 nm

Dissolved oxygen (DO) vs Bilirubin content

Dissolved oxygen (DO) is largely consumed by the components in the closed

container after 14 days. Bilirubin content is stabilized after the dissolved oxygen

is removed after 14 days at 45 C.

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

Day 0 Day 7 Day 14 Day 21 Day 30

Abso

rbance (

459 n

m)

Mean DO BIL 459nm

1.00

0.80

0.40

0.00

0.20

DO

(m

g/L)

0.60

Prediction of the shelf life with ASAP data

according to the Q10 Rule

Shelf Life Current Unsparged Overfilled

ASAP at 45C

(Days)14 days 7 days 21 days

Projected for 5C

(Months)

224 days = 7.4 mo

(RTS 7)

112 days = 3.7 mo

(RTS 4)>336 days = 11 mo

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

Day 0 Day 7 Day 14 Day 21 Day 30 Day 60 Day 90 Day 120

Test Case #6 Unsparged 45C

Mean 4.0 mg/dL 2.0 mg/dL

] Acceptance criteria

Reflecta

nce

of B

IL(%

)

Q10 = 2

Q40 = 24=16

Preliminary study conclusions with ASAP

45C stress is informative of product stability:

Current conditions showing initial failures in the 14-21 day range

Unsparged failed at 7 days

Overfill was stable to 21 days

35C is expected to produce more differentiation but may take longer

These results suggest that increasing filling volume is the best approach

Summary of the workshop on ASAP

Stability studies can be complex and need science-based decisions

ASAP can provide estimates of the kinetic parameters for the rates of

decompositions

The results of ASAP can be used to characterize the relationship between the

degradation and storage conditions

The results supply critical information in the design and analysis of long-term

stability studies under ambient and refrigerated conditions

All studies require detailed information about the products to establish the end

of shelf-life parameters

Follow guideline, use standard model to predict shelf-life claims