Design and Implementation of Forced Degradation/Chemical Stress Studies using HPLC

Dr. Michael Swartz, Ph. D.

Director of R&D, Analytical Chemistry Synomics Pharma Services

mswartz@synomicspharma.com

Synomics 2010

Presentation Outline

Forced Degradation/Chemical Stress Study Definition

How and why they are used

Stability Indicating Methods

Method development and validation

Newer Column and Detector Technology Case Study Examples Available Guidance and References

Synomics 2010

Synomics Pharma Services

Part of the Smithers Group
Established in 1925 Dedicated to contract research servicing pharmaceutical, environmental, agrichemical, polymer, and automotive markets Over 500 employees in Europe (England, Switzerland), Japan, and the US (OH, NC, TX, MA)

Massachusetts Business Unit with > 100 employees

Springborn Laboratories established in 1969 Synomics Pharma Services spun-off from Springborn in 2004
Fully FDA Compliant and Inspected GLP/GMP Facility Focus on Pharmaceutical, biotech and consumer products industries

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Forced Degradation (Chemical Stress) Studies

What is a Forced Degradation Study?
Study designed to intentionally degrade a drug substance or drug product Heat, light, moisture, pH

Why Do Forced Degradation Studies?

Understand the reactive chemistry of the drug substance Help anticipate future stability issues of both drug substance and drug product Provides useful information for formulation and stability May be required for regulatory submissions Generate a sample for development of stability indicating methods for formal stability study support

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Example Conditions for Forced Degradation

STUDY Acidic pH Neutral pH Basic pH Oxidation Photolysis (UV) Photolysis (Fluorescence) CONDITIONS 0.1N HCl pH 7.0 Phosphate Buffer 0.1N NaOH 02 Atmosphere, or H2O2 1000 Watt h/M2 6x106 lux h

Goal: Degrade API 5-10%; Stability Indicating Method (SIM)

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What is a Stability Indicating Method?

A Stability Indicating Method (SIM) Is:

A validated method that can accurately and precisely quantitate the decrease of the API content due to degradation Is specific for the drug substance Shows a decrease in assay value (correlated to drug substance loss) due to degradation Has no interference from excipients, impurities or degradation products Detects and quantitates impurities and degradation products

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Why Are Stability Indicating Methods Needed?

To support long term stability testing

Demonstrate how the quality of the drug substance or product changes over time in response to environmental factors Temperature Humidity Light Establishes storage and packaging conditions

Its good science, and Its the law

CFR Title 21, Section 211

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When to Use Stability Indicating Methods

When Are SIMs Needed?
Stability Studies API release Drug product release Toxicology dosing solutions Excipient compatibility and pre-formulation Packaging studies

When Are SIMs Not Needed?

In process controls Secondary assay for API Titration Inorganics

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The Path to a Stability Indicating Method

Three Steps Generating the Sample Developing the Method Validate the Method

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SIM Method Development Challenge

W1_COL1_70_S2
W1_COL1_70_S3 100

JNJ27387581_RSV

08-Mar-2006 17:18:33
2: Diode Array TIC 1.50e7

W1_COL1_70_S3
W1_COL1_70_S3 100

JNJ27387581_RSV

08-Mar-2006 17:54:28
2: Diode Array TIC 1.50e7

Excipients

-20 W1_COL1_70_S2 100

Impurities

2: Diode Array TIC 3.87e6

-19 W1_COL1_70_S1 100

Degradants

2: Diode Array TIC 1.50e7

Composite sample
-20 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Time -20 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Time

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Courtesy of: Dr. Rudy Sneyers, J&J Belgium

Stability Indicating Methods

Method Development Issues and Challenges

10

Same as Method Development in General

Additional requirements Resolution UV Sensitivity LOQ Column temperature control is critical

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SIM Method Development Approaches

Trial and Error Literature Study

Standard method?

Experience Software Assisted Method or Column Scouting

Further Optimization

Goals: #1-No Co-elutions with DS #2-Baseline Resolution of all Analytes

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Expanding Range of LC Selectivity MeOH High

Method Goals

Solvents
MeCN C8

pH
Low Phenyl

C18
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RP18

Columns

Reversed Phase Chromatography

Polar Modifier Choices

13

Reversed-Phase Retention Map

40 35 Retention Factor (k) 30 25 20 15 10 5 0 0

Note: Retention of neutral analytes not affected by pH

Acid
Increased acid retention

Neutral
Increased, robust base retention

Note: Column Particle, Temperature and % Organic Held Constant

Base
2 4 6 pH 8 10 12

Silica pH Range Hybrid Particle pH Range

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Why Validate?

Method validation is completed to insure that an analytical methodology is accurate, reproducible and rugged over the specific range that an analyte will be analyzed. Method validation provides assurance of reliability. FDA Compliance

"The process of providing documented evidence that something does what it is intended to do."

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The Validation Process

System Suitability Method Validation Analytical Instrument Qualification Software Validation

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USP 32 NF27 Chapter 1225 Analytical Performance Characteristics

Specificity Accuracy Linearity

16

Method Validation

Range Precision Limit of Quantitation Limit of Detection Robustness

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Stability Indicating Methods Must be Validated

Validation Characteristics Versus Type of Analytical Method

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Analytical Category 2: Impurities Category 3: Category 4: Category 1: Performance Quant. Limit Tests Specific Tests I.D. Assays Parameter Accuracy Precision Specificity LOD LOQ Linearity Range Robustness Yes Yes Yes No No Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes * No Yes Yes No No No No * Yes * * * * * Yes No No Yes No No No No No

* May be required, depending on the nature of the specific test.

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Stability Indicating Method Critical Validation Parameters

Specificity Accuracy Linearity

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Method Validation

Range Precision Limit of Quantitation Limit of Detection Robustness

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Specificity (Selectivity)

Separation
Resolution Determination of separation between peaks Plate Count Determination of a systems efficiency Tailing Factor Calculation referencing peak shape

No Interference at API Retention Time

Blanks Placebo

Spectral Information
PDA MS

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Changes Courtesy of ICH: Specificity

20

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Recent FDA Comments-Peak Purity

The purity testing provided was based on one wavelength and on one lot. This is not adequate to assess peak purity. Typically, the entire UV-Vis spectra (with consideration for solvent and noise controls) is analyzed at various points in the peak area (especially at leading and trailing edge) and at varying concentrations to indicate purity. There is chromatographic software to perform this type of analysis. In addition, other instruments and techniques are used to assess peak purity. We are unable to fully evaluate the purity of the compound based solely on the information provided.

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Role of Photodiode Array Detector in LC Method Development/Validation

22

Spectral Contrast Software For Peak Tracking and Identification Potential For Peak Purity System Suitability Criteria Peak Purity or Homogeneity Information Indicative of CoElutions What is Needed for PDA Peak Purity Calculations?
Compounds must have UV Absorbance Some Degree of Chromatographic Resolution Some Degree of Spectral Differences Between Compounds

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PDA Spectral Contrast Theory

Comparison of the two vectors A and B - calculation of angle If A and B totally different, = 90 If A and B totally identical, = 0

23

AU

nm
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Optimized Separation?

Peak 1

Peak 2

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PDA Peak Purity Plot for Peak 2

Purity Angle: 0.064 Purity Threshold: 0.261

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PDA Peak Purity Plot for Peak 1

Purity Angle: 4.224 Purity Threshold: 0.278

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Detection and Quantitation Limit

Visual (Non-Instrumental Methods) Signal To Noise Ratio (3 or 2:1 and 10:1 is Typical) Limit may be based on the standard deviation of the response and slope:

Limit = (K)STD/S
K = 3.3 for LOD, 10 for LOQ STD = standard deviation of the response S = slope of the calibration curve
Both DL and QL are validated by analyzing a suitable number of samples at the limit. Method should be documented.
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ICH Q3A(R2)-Thresholds for Impurities in New Drug Substances

28

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HPLC Columns and Effect on LOQ

0.0005

A) Brand A C18 Waters Symmetry C18 Signal to Noise = 11

AUFS

5.00

Minutes

10.00

0.0005

B) Brand X C18 Signal to Noise = 6.5

AUFS

Sample: 0.25 g/mL Tamoxifen Injection volume and linear flow velocity compensated for on both columns

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5.00

Minutes

10.00

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Robustness: Definition

Robustness
Measure of the capacity to remain unaffected by small (deliberate) variations in method parameters Indication of reliability during normal use Evaluate during method development Used to set system suitability specifications

Robustness Ruggedness

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Robustness Study Design

Univariate Versus Multivariate Screening Designs Used in method validation

Full factorial Fractional Factorial Plackett-Burman

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Full Factorial

Every Possible Combination of Factors Results in 2k runs

Four factors results in 16 runs Nine factors results in 512 runs!!

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Full Factorial Experimental Design For Four Factors

% Organic

33

Wave length

Wave length

Flow

Flow

pH

pH

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Fractional Factorial Design

Carefully chosen fraction or subset of factor combinations

Degree of fractionation: Scarcity of effects principle , , etc. E.G. Nine factors 1/16 fraction = 32 runs

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Fractional Factorial Design For Five Factors

% Organic

35

Wave length

Wave length

Flow pH pH % Organic

Flow

Column Temp

Wave length

Wave length

Flow pH
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Flow pH

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Plackett-Burman Design
Looks at only main effects Experiments in multiples of four rather than powers of two
Run 1 2 3 4 5 6 7 8 9 10 11 12 Pattern ++++++++++++ -+-+++---+--+-+++---+ +--+-+++---+--+-+++---+--+-+++---+--+-+++ ++---+--+-+ +++---+--++++---+--+-+++---+--+ +-+++---+-X1 1 -1 -1 1 -1 -1 -1 1 1 1 -1 1 X2 1 1 -1 -1 1 -1 -1 1 1 1 1 -1 X3 1 -1 1 -1 -1 1 -1 -1 1 1 1 1 X4 1 1 -1 1 -1 -1 1 -1 -1 -1 1 1 X5 1 1 1 -1 1 -1 -1 -1 -1 -1 -1 1 X6 1 1 1 1 -1 1 -1 1 -1 -1 -1 -1 X7 1 -1 1 1 1 -1 1 -1 1 1 -1 -1 X8 1 -1 -1 1 1 1 -1 -1 -1 -1 1 -1 X9 1 -1 -1 -1 1 1 1 1 -1 -1 -1 1 X10 1 1 -1 -1 -1 1 1 -1 1 1 -1 -1 X11 1 -1 1 -1 -1 -1 1 1 -1 -1 1 -1

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Determining the Factors: Isocratic Method

Factor Organic Solvent Concentration Buffer Concentration Buffer pH (if adjusted) Temperature Flow rate Wavelength Injection Volume Limit Range (+/-) 2-3% 1-2% 0.1-0.2 pH units 3C 0.1-0.2 mL/min. 2-3nm for 5nm bandwidth Injection type and size dependant

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These limits are examples only and should be chosen according to expected laboratory and instrument variations.

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Determining the Factors: Gradient Method

Factor Initial hold time* Slope and length Limit Range (+/-) 10-20 % of segment time The slope is set by the initial %B and the final %B, as well as the gradient length. It is recommended to adjust the lengths by 10-20% and allow the slope to vary.

38

Final hold time

Adjusted according to the last eluting compound and varied accordingly

Factors and limits listed here are in addition to many of the factors considered in an isocratic method. *It is increasingly common for gradient methods to have an initial hold time to accommodate transfer to instruments with different dwell volumes.
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Case Study #1: Original Method Not Stability Indicating

39

Challenge:
Insufficient Resolution in Photostability Experiments Isnt MS Compatible

Solution:
UHPLC Rapid Method Re-Development Scale-up to HPLC

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Existing HPLC Method

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UHPLC MS Compatible Method

41

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UHPLC MS Compatible Method, 100mm Column

42

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Scaled-up HPLC Method

MS Compatible, 150mm Column, 35 Minute Cycle Time

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Case Study #2: Forced Degradation

Simvastatin Drug Substance Adapting Typical Forced Degradation Conditions Is Method Stability Indicating?
New Column Technology Buffer Change for MS Compatibility

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USP HPLC Method Conditions

Waters Alliance HPLC System with a 2996 PDA and a 2487 Variable Detector monitoring 238 nm
Columns Waters Xbridge C18, 5m, 4.6 x 250 mm Halo Fused-Core C18, 5m, 3.0 x 75 mm 45 C MPA: 15 mM Phosphate pH 4.5 MPB: ACN Flow USP-1.5 mL/min Halo-0.6 mL/min Injection volume USP-40 L Halo-5 L

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New Column Technology

Halo Fused-Core Particles

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Figures courtesy of Mac-Mod Analytical Inc.

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Column Comparison

USP Column

Halo Column

35/65 Phosphate/ACN

35/65 Phosphate/ACN

Injection Volume scaled from 40 (USP) to 5 L (Halo) Halo column: 3.0 x 75 mm, 0.6 mL/min.
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MS Compatible Buffer
Stability Indicating?
USP Column Halo Column

48

35/65 Acetate/ACN

35/65 Acetate/ACN

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MS Compatible Buffer-Decrease Organic

USP Column

Halo Column

45/55 Acetate/ACN

45/55 Acetate/ACN

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Actual Initial Forced Degradation Conditions

Procedure ID Drug Substance Reagent #1 Condition Immediately diluted to the final concentration 24 hrs. room temperature 24 hrs. room temperature Reagent #2 Diluent 4.5 mL of Diluent per HPLC Method 2.5 mL of Diluent per HPLC Method 2.5 mL of Diluent per HPLC Method 3.0 mL of Diluent per HPLC Method 3.5 mL of Diluent per HPLC Method 4 mL of Diluent per HPLC Method 4.5 mL of Diluent per HPLC Method 9 mL of Diluent per HPLC Method 9 mL of Diluent per HPLC Method Final Conc.

Drug Substance Control

0.5 mL of 5 mg/mL API Stock Solution 0.5 mL of 5 mg/mL API Stock Solution 0.5 mL of 5 mg/mL API Stock Solution

N/A

N/A

0.5 mg/mL

Acidic Solution

Add 1.0 mL 0.1N HCl Add 1.0 mL 0.1N NaOH Add 1.0 mL 0.1N HCl Add 1.0 mL 3% H2O2 Add 1.0 mL of 3% H2O2 N/A

Add 1.0 mL 0.1N NaOH Add 1.0 mL 0.1N HCL Add 1.0 mL 0.1N NaOH

0.5 mg/mL

Basic Solution

0.5 mg/mL

Acid/Base Control

NA

NA

0.5 mg/mL

Oxidative Solution

0.5 mL of 5 mg/mL API Stock Solution

24 hrs. room temperature

N/A

0.5 mg/mL

Oxidative Control

N/A

N/A

N/A

0.5 mg/mL

Heat

0.5 mL of 5 mg/mL API Stock Solution 1.0 mL of 5 mg/mL API Stock Solution 1.0 mL of 5 mg/mL API Stock Solution

24 hrs at 60C

N/A

0.5 mg/mL

Light #1

NA

1X ICH Q1B

N/A

0.5 mg/mL

Light #2

NA

3X ICH Q1B

N/A

0.5 mg/mL

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Adapting Typical Forced Degradation Conditions

51

0.1 N NaOH

0.015 N NaOH

Goal: 5-10% Degradation

0.030 N NaOH

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Example Forced Degradation Chromatograms

Acid 1X ICH

Base

3X ICH

Heat

Peroxide

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Case Study #3: Adrenocorticotropic Hormone Fragment 4-10 Peptide

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Hepta-Peptide
Seven discreet AAs with no repeats MW 962.1

Peptide Stability
AA or peptide fragment degradants

Amino Acid Analysis

Acid Hydrolysis

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Peptide Separation Conditions

Mobile Phase: 0.1% TFA in water (A) and ACN (B) Column: C18 4.6 x 250 mm 5m Column Temperature: 30 C Flow Rate: 0.6 mL/min. Gradient: 5 min hold at 100% A, 0-40% B over 20 min. Sample: 1 mg/mL Injection Volume: 10 L Detection
UV CAD

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Corona Charged Aerosol Detector (CAD)

Highest Sensitivity of Universal Type Detectors Wide Dynamic Range Detects any Non-volatile or Semi-volatile Compound
No chromophore required

Consistent Reproducible Response Easy Intuitive Operation

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Corona CAD Under the Hood

The liquid eluent from the HPLC column enters the Corona (1) where it undergoes pneumatic nebulization by nitrogen or air (2). Small droplets enter the drying tube (3) and form particles while large drops exit the drain (4) to waste.
1 3 2 10

8 4 6

Dried particles enter the mixing chamber (5). Another gas stream passes 5 7 over a charged Corona Needle (6). Charged gas High mobility species are removed by an ion trap (8) while the then mixes with the dried remaining charged particles pass to a collector where the particles forming charged charge is measured with a very sensitive electrometer (9). particles (7). Signal is transferred to chromatographic data software (10).
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RP-HPLC Peptide Separation: CAD/UV Detection

300.00

57

Corona CAD

mV

200.00

100.00

0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Minutes 16.00 18.00 20.00 22.00 24.00 26.00 28.00

0.80

0.60 AU

UV 214 nm

0.40

0.20

0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Minutes 16.00 18.00 20.00 22.00 24.00 26.00 28.00

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RP-HPLC AA Separation: CAD/UV Detection

Corona CAD
Met
50.00 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Minutes 16.00 18.00 20.00 22.00 24.00 26.00 28.00

250.00 200.00 mV 150.00 100.00

Phe

Trp

His Gly

Arg

Glu

2.50 2.00 1.50 1.00 0.50 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Minutes 16.00 18.00 20.00

Trp

AU

His

UV 214 nm
Phe

22.00

24.00

26.00

28.00

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Peptide Forced Degradation

1N HCl at 110C, 6 hours

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0.60 0.50

UV 280 nm
0.40 0.30 0.20 0.10 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Minutes 16.00 18.00 20.00 22.00 24.00 26.00 28.00

~ 5 Peaks

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AU

Peptide Forced Degradation

1N HCl at 110C, 6 hours

60

2.00

UV 214 nm
~ 12 Peaks Variable Response

1.50 AU 1.00 0.50 0.00 0.00 2.00 4.00

6.00

8.00

10.00

12.00

14.00 Minutes

16.00

18.00

20.00

22.00

24.00

26.00

28.00

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Peptide Forced Degradation

1N HCl at 110C, 6 hours

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400.00

CAD
300.00

>14 peaks Low Response Variation

mV

200.00

100.00

0.00

2.00

4.00

6.00

8.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 Minutes

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Guidance and References

ICH Q1A: Stability Testing of New Drug Substances and Products. (International Conference on Harmonization of Technical Requirements for the Registration of Drugs For Human Use, Geneva, Switzerland, February 2003). See also at www.ICH.org. ICH Q1B: Stability Testing: Photostability Testing of New Drug Substances and Products (International Conference on Harmonization of Technical Requirements for the Registration of Drugs For Human Use, Geneva, Switzerland, November 1996). See also at www.ICH.org. Guideline for submitting samples and analytical data for methods validation. Food and Drug Administration, February 1987. US Government Printing Office:1990-281-794:20818. Guidance for Industry, Analytical Procedures and Method Validation, U.S. Department of Health and Human Services FDA, August 2000. ICH Q2 (R1) Validation of Analytical Procedures: Text and Methodology Reynolds, D. W. etal, American Pharm. Review, May/June, 56-61 (2004). Alsante, K. M. etal, Advanced Drug Delivery Reviews 59 (2007) 2937. Pharmaceutical Stress Testing, Predicting Drug Degradation, Baertschi, S. W. Editor, Informa Healthcare USA Inc., New York, 2007.

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Presentation Outline

Forced Degradation/Chemical Stress Study Definition

How and why they are used

Stability Indicating Methods

Method development and validation

Newer Column and Detector Technology Case Study Examples Available Guidance and References

Synomics 2010

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Its all about Risk..

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Acknowledgements

Colleagues @ Synomics Mark Emanuele Amber Awad Adam Grenier Debby Hartley Dionex/ESA Bruce Bailey Christopher Crafts Ian Acworth

And:

mswartz@synomicspharma.com

Synomics 2010