mswartz@synomicspharma.com
Synomics 2010
Presentation Outline
Newer Column and Detector Technology Case Study Examples Available Guidance and References
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Three Steps Generating the Sample Developing the Method Validate the Method
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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
Impurities
Degradants
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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Solvents
MeCN C8
pH
Low Phenyl
C18
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RP18
Columns
13
Acid
Increased acid retention
Neutral
Increased, robust base retention
Base
2 4 6 pH 8 10 12
14
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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Method Validation
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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
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Method Validation
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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
Spectral Information
PDA MS
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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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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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AU
nm
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Optimized Separation?
Peak 1
Peak 2
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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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0.0005
5.00
Minutes
10.00
0.0005
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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Full Factorial
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Wave length
Wave length
Flow
Flow
pH
pH
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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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These limits are examples only and should be chosen according to expected laboratory and instrument variations.
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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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Challenge:
Insufficient Resolution in Photostability Experiments Isnt MS Compatible
Solution:
UHPLC Rapid Method Re-Development Scale-up to HPLC
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Simvastatin Drug Substance Adapting Typical Forced Degradation Conditions Is Method Stability Indicating?
New Column Technology Buffer Change for MS Compatibility
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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
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35/65 Acetate/ACN
35/65 Acetate/ACN
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USP Column
Halo Column
45/55 Acetate/ACN
45/55 Acetate/ACN
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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
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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0.1 N NaOH
0.015 N NaOH
0.030 N NaOH
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Base
3X ICH
Heat
Peroxide
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Hepta-Peptide
Seven discreet AAs with no repeats MW 962.1
Peptide Stability
AA or peptide fragment degradants
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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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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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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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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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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
60
2.00
UV 214 nm
~ 12 Peaks Variable Response
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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400.00
CAD
300.00
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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Presentation Outline
Newer Column and Detector Technology Case Study Examples Available Guidance and References
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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