1. Pharmaceutical Development with Focus on Paediatric formulations
WHO/FIP Training Workshop
Hyatt Regency Hotel
Sahar Airport Road
Andheri East, Mumbai, India
28 April 2008 – 2 May 2008

2. Analytical Method Development
Presented by:
Birgit Schmauser, PhD
Federal Institute for Drugs
and Medical Devices (BfArM)

3. Analytical Method Development
In this presentation:
Standards in developing analytical methods for
Originator and multisource generic FPPs
Specifications
Stability
Parallel development of analytical methods for cleaning validation

4. Analytical Method Development
Originator, First-time Generic and Multisource Generic
Multisource Generic
First-time Generic
Originator
Pharmacopoeias
Information from regulatory agencies (publicly available) & literature data
Originator´s specifications
API quality standards
FPP quality standards
Verify identity, potency, purity of API and FPP by pharmacopoeial methods and in-house methods
Derive identity, potency, purity of API and FPP by in house methods
Establish identity, potency, purity of API and FPP by in-house methods
Analytical methods

5. Analytical Method Development
HPLC-method to assay potency and purity – risk assessment
Multisource Generic
First-time Generic
Originator
Selectively screen/detect any impurity or degradant Establish potency
Identify impurities/degradants
Verify impurities from Pharmacopoeia Characterise „in-house“ impurities/degradants (Response factors)
Derive impurities/degradants from Originator Characterize „in-house“ impurities/degradants Calculate response factors
Characterise all impurities/degradants Calculate Response factors (qualification by clinical use)
Use pharmacopoeial reference materials
Extract (& reproduce) reference materials
Establish reference materials
„Implement“ for routine use
Adapt/modify to/for routine use
Adapt to routine use

6. Analytical Method Development
Interchangeability (IC) of multisource generic FPPs
(Essential similarity with Innovator FPP)
Pharmaceutical + Bioequivalence Equivalence
IC = PE + BE

7. Analytical Method Development
Pharmaceutical equivalence
FPPs meet the same or comparable standards by use of equivalent analytical methods
Same API (chemical and physical equivalence)
Same dosage form and route of administration
Same strength
Comparable labeling
Equivalence in pharmaceutical development
Equivalence in stability
Equivalence in manufacture (WHO-GMP)

8. Analytical Method Development
Prequalification requirements
Validation of analytical methods is a prerequisite for prequalification of product dossiers
Non-compendial APIs and FPPs are tested with methods developed by the manufacturer
For compendial APIs and FPPs the „applicability“ of pharmacopoeial methods to particular products must be demonstrated (verification)
Analytical methods must be developed and validated according to TRS 823, Annex 5, Validation of analytical procedures used in the examination of pharmaceutical materials ; ICH Q2 (R1)
To be used within GLP and GMP environments

9. Analytical Method Development
Use of analytical methods - generics
METHODS
PHARMACEUTICAL
CLINICAL
At initial phase of pharmaceutical development
To understand the profile of related substances and to study stability
To start measuring the impact of key product and manufacturing process parameters on consistent FPP quality
To develop a stable and reproducible formulation for the manufacture of bioequivalence, dissolution, stability and pilot-scale validation batches
To determine bioavailability in healthy volunteers
At advanced phase of pharmaceutical development
To be robust, transferable, accurate and precise for specification setting, stability assessment and QC release of prequalified product batches
To optimise, scale-up and transfer a stable and controlled manufacturing process for the prequalification product
To prove bioequivalence after critical variations to the prequalified dossier

10. Analytical Method Development
Prerequisites for analytical method validation
Six “M”s
Quality of the analytical method
Man
Machine
qualified
calibrated
robust
Methods
suitable
characterised
documented
Milieu
Material
Management
Quality
Reference standards
Tempe- rature
Analysts´ support
skilled
Humidity
Vibrations
Time
Supplies
Irradi- ations

11. Analytical Method Development
Method development life cycle
Method development
Initital Method Development
Pre-Validation Evaluation
Method Optimization
Robustness
System Suitability
Planning
Development Plan – Project
Development and Validation Policy
Objectives/Requirements of Method
Information Gathering
Resource Gathering
Validation Experiments
Customer Evaluation Testing
Periodically Monitoring/Review of Methods in Control Labs
Method Transfer Experiments
Filed Method in Use
From: Analytical Chemistry in a GMP Environment. Edited by J.M. Miller and J.B. Crowther, ISBN , Wiley & Sons Inc.

12. Analytical Method Development
Validation should verify the suitability of an analytical method for its intended purpose
Validation should be founded on method development performed beforehand that suggest the suitability and robustness of the method
Validation may be performed in different ways (individual purpose) according to common standards

13. Validation protocol
Method principle / objective
Listing of responsibilities
Laboratories involved and their role in the validation
Method categorization
List of reagents (including test lots) and standards
Test procedures to evaluate each validation parameter and proposed acceptance criteria
Plan or procedure when acceptance criteria are not met
Requirements for the final report
The validation process cannot proceed until the protocol and all parties involved approve the acceptance criteria

14. Analytical Method Development
Innovator versus Generics
Generics
Innovator - +
R & D on API
Preclinical trials
Method validation summary
Clinical trials phase I and II
Method validation completed
Clinical trials phase III
Validated methods
Post marketing phase IV
Validated methods: GMP and GLP
Entering of Generics; Pharmaceutical development, Comparability with Innovator

15. Analytical Method Development
Validation Characteristics
Assay
Impurities
Identification
limit
quantitative + -
Accuracy
Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range
Robustness

16. Analytical Method Development
Accuracy and precision
Accurate & precise
Accurate & imprecise
Inaccurate &
precise
Inaccurate & imprecise

17. Analytical Method Development
Precision
Expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample
Is usually expressed as the standard deviation (S), variance (S2) or coefficient of variation (RSD) of a series of measurements
Precision may be considered at three levels
Repeatability (intra-assay precision)
Intermediate Precision (variability within a laboratory)
Reproducibility (precision between laboratories)

18. Analytical Method Development
Normal distribution, probability function [P(x)] and confidence interval [CI]
Probability (P), that measurements from a normal distribution fall within [µ-xn, µ+xn] for xn = ns is described by the “erf-function” (µ = mean):
An interval of ± 3 s covers 99.73% of values P xn s 2s 3s 4s 5s
Number of times each value occurs s
Values s 3s 2s 2s 3s

19. Analytical Method Development
Normal distribution, probability function [P(x)] and confidence interval [CI]
Probability-P Confidence interval [CI] centered around the mean [µ] in units of sigma [s] described by “inverse erf-function”:
A CI of 95% includes values ± 1.95 s around the mean xp P s
0.800 s
0.900 s
0.950 s
0.995 s
0.999

20. Analytical Method Development
Relationship of variability, probability and reliability of data
High variability of data (large s) generate large confidence intervals and thus lower the reliability of the mean
Low variability of data (small s) generate small confidence intervals and thus increase the reliability of the mean

21. Analytical Method Development
Repeatability
Six replicate sample preparation steps from a homogenously prepared tablet mixture (nominal value of API 150 mg)
Assay
Peak area
Injection
mg/98.06%
173865 1
mg/98.66%
174926 2
mg/97.54%
172933 3
mg/98.72%
175011 4
mg/101.30%
179557 5
mg/99.52%
176425 6
mg/98.96%
175453
Mean
1.98 mg/1.32%
2329
SD (s)
1.32%
RSD
Mean ± 3 SD = Confidence interval of 99.73%
98.96 ± 3x1.32% = 95% %

22. Analytical Method Development
Intermediate precision
Expresses within-laboratories variations (different days, different analysts, different equipment etc.)
Peak area analyst 3
Peak area analyst 2
Peak area analyst 1
Injection
177965
175656
173865 1
178556
175878
174926 2
177342
176004
172933 3
178011
176344
175011 4
179466
175332
179557 5
179688
174959
176425 6
178504
175695
175453
Mean
918
495
2329
SD (s)
0.51%
0.28%
1.32%
RSD
Mean ± 3 SD: (  100%)
Analyst 1: 98.96% ± 3 x 1.32%
Analyst 2: 99.12% ± 3 x 0.28
Analyst 3: % ± 3 x 0.51
Average of 3 analysts ± 3SD: 95% %

23. Analytical Method Development
Reproducibility
Expresses the precision between laboratories
Collaborative studies, usually applied to standardisation of methodology
Transfer of technology
Compendial methods

24. Analytical Method Development
Accuracy
Expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found
Sometimes referred to as „TRUENESS“
true
mean

25. Analytical Method Development
To find out whether a method is accurate:
Drug substance (assay)
Application of the method to an analyte of known purity (e.g. reference substance)
Comparison of the results of one method with those of a second well-characterised method (accuracy known)
Drug product (assay)
Application of the method to synthetic mixtures of the drug product component to which known quantities of the analyte have been added
Drug product may exceptionally be used as matrix
Drug substance/Drug product (Impurities)
Application of the method to samples spiked with known amounts of impurities

26. Analytical Method Development
Accuracy: Application of the method to synthetic mixtures of the drug product components to which known quantities of the analyte have been added
Recovery reduced by ~10 – 15%
From: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman Lam, Y.C. Lee and Xue-Ming Zhang, ISBN , Wiley & Sons

27. Analytical Method Development
When to expect Accuracy problems
Insufficient selectivity of the method
Impurity peaks are not resolved and account for assay value
Recovery is < 100%
Irreversible adsorption of analyte to surfaces of the system
Incorrect assay value of a reference standard
Due to decomposition of reference standard
Incorrect assay value due to change in matrix
Analytical laboratory still uses the preceding matrix as standard

28. Analytical Method Development
Specificity
Is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present (impurities, degradants, matrix…)
Identity testing
To ensure the identity of an analyte
Purity testing
To ensure accurate statement on the content of impurities of an analyte
Assay
To allow an accurate statement on the content of an analyte in a sample

29. Analytical Method Development
Specificity: Overlay chromatogram of an impurity solution with a sample solution
From: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman Lam, Y.C. Lee and Xue-Ming Zhang, ISBN , Wiley & Sons

30. Analytical Method Development
Specificity and stability
Stress stability testing to ensure the stability indicating potential of an analytical method
Apply diverse stress factors to the API
Apply diverse stress factors to the FPP
Stress conditions: e.g. Supplement 2 of Generic Guideline; TRS 929, Annex 5
Assure that the API can be assessed specifically in the presence of known and unknown (generated by stress) impurities
Assure that known impurities/degradants can be specifically assessed in the presence of further degradants
By peak purity assessment and (overlay of) chromatograms

31. Analytical Method Development
Stress stability studies versus forced degradation studies
Stress stability (5 – 15% decomposition)
Forced degradation
Stress parameter
pH ± 2 (2 weeks)
0.2 ml 1N HCl / 5 ml API-solution / 3h, 6h, 12h, 24h…7d (RT & 60°C)
Acid
pH ± 10 (2 weeks)
0.2 ml 1N NaOH / 5 ml API-solution / 3h, 6h, 12h, 24h…7d (RT & 60°C)
Base
1 g/ml oxygen bubbled through (8 hours) 0.1 – 2% H2O2 (24 hours)
0.2 ml 5% or 35% H2O2 / 5 ml API- solution (RT, to 7d & 60°C, 3h)
H2O2 / Oxygen -
60°C / 5 ml solution (3h, 6h…7d)
Heat
60°C (4 weeks)
105° C / solid API (1d and 7d)
365 nm or white fluorescent light / solid API (1d and 7d)
UV or Light
50°C / 80% RH (4 weeks)
Humidity

32. Analytical Method Development
Limit of Detection (LOD, DL)
The LOD of an analytical procedure is the lowest amount of analyte in sample which can be detected but not necessarily quantitated as an exact value
Determination is usually based on
Signal to noise ratio (~3:1) (baseline noise) or
Standard deviation of response (s) and Slope (S)
3.3 s/S

33. Analytical Method Development
Limit of Quantitation (LOQ, QL)
The LOQ is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy
The quantitation limit is used particularly for the determination of impurities and/or degradation products
Determination is usually based on
Signal to noise ratio (~10:1) (baseline noise) or
Standard deviation of response (s) and Slope (S)
10 s/S

34. Analytical Method Development
LOD, LOQ and Signal to Noise Ratio (SNR)
LOQ
Signal to Noise = 10:1
LOD
Signal to Noise = 3:1
Noise

35. Analytical Method Development
LOQ
Quantitation by SNR is accepted
Quantitation by Standard deviation of response (s) and Slope (S) (10 s/S) is more adequate as it involves the response of the actual analyte
Best to calculate in the region close to y-intercept

36. Analytical Method Development
LOQ and impurities
In determination of impurities in APIs and FPPs the LOQ should be determined in the presence of API
LOQ should be NMT reporting level
LOQ should be given relative to the test concentration of API
Specificity of impurity determination should always be demonstrated in the presence of API at API specification levels
Spiking of test concentration (API/FPP) with impurities at levels of their specification range

37. Analytical Method Development
Spiking
API test concentration (normalised)
0.1 mg/ml (100%)
Impurity spiking concentrations
0.001 mg/ml (1%) – specification limit
mg/ml (0.1%) – limit of quantitation (minimum requirement)
API at test concentrations
API below test concentrations

38. Analytical Method Development
Linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample
If there is a linear relationship test results should be evaluated by appropriate statistical methods
Correlation coefficient (r)
Y-intercept
Slope of regression line
Residual sum of squares
PLOT OF THE DATA

39. Analytical Method Development
Usual acceptance criteria for a linear calibration curve
r > 0.999; y-intercept a < 0 to 5% of target concentration RSD (wrt calibration curve) < 1.5-2%
r > 0.997
r < 0.997
From: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman Lam, Y.C. Lee and Xue-Ming Zhang, ISBN , Wiley & Sons

40. Analytical Method Development
Range
The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity

41. Analytical Method Development
Range
Assay
80 to 120% of test concentration
Content uniformity
70 to 130% of test concentration
Dissolution
Q-20% to 120%
Impurities
Reporting level – 120% of specification limit (with respect to test concentration of API)
Assay & Impurities
Reporting level to 120% of assay specification

42. Analytical Method Development
Linearity is limited to 150%of shelf life specification of impurities
Test concentration can be used to determine impurities
To determine drug substance (assay) the test concentration must be diluted
The range is 0 – ~ 150% of impurity specification
From: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman Lam, Y.C. Lee and Xue-Ming Zhang, ISBN , Wiley & Sons

43. Analytical Method Development
Robustness
Robustness of an analytical procedure should show the reliability of an analysis with respect to deliberate variations in method parameters
The evaluation of robustness should be considered during the development phase
If measurements are susceptible to variations in analytical conditions the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure

44. Analytical Method Development
Influence of buffer pH and buffer concentration in mobile phase on retention times of API and impurities
Conclusion: The buffer composition should be maintained in a range of 85 ± 0.5%
Missing: Acceptance criterion for maximal deviation of retention time should be defined unless justified
Impurity C
Impurity B
Impurity A
API
8.26
7.43
3.86
10.46
As is
8.38
7.51
3.94
10.45
buffer pH 5.9
8.34
7.49
buffer pH 6.9
6.66
6.16
3.43
7.84
Buffer conc. 83%
11.18
9.61
4.77
15.26
Buffer conc. 87%

45. Analytical Method Development
System suitability testing
Based on the concept that equipment, electronics, analytical operations and samples to be analysed constitute an integral system that can be evaluated as such
Suitability parameters are established for each analytical procedure individually
Depend on the type of analytical procedure

46. Analytical Method Development
Method stability
System suitability over time
Sample solution stability
A solution of stavudine is stable for ~ 2 h, then it starts to degrade to thymine
Impurity-spiked sample solution stability
A solution containing stavudine spiked with its impurity thymine does not allow to clearly distinguish between degradation and spike
A solution containing stavudine of a FPP-stability sample solution does not allow to clearly distinguish between FPP-stability degradation and sample solution degradation
Should be analysed immediately

47. Analytical Method Development
When to be „surprised“ about validation data:
Precision of impurity determination
Precision of API determination
Method precision of released API (dissolution)
% RSD 0.33 – 2.25
System precision
% RSD 0.0
Method precision
% RSD 0.08
Average peak area
% RSD ≤ 2.0
Acceptance criterion
% RSD 0.4
Average peak area
% RSD ≤ 10.0
Acceptance criterion

48. Analytical Method Development
Specification range (USL-LSL)
Process variability (usually ± 2 SD)
Analytical variability (± 3s)
~ NMT 30% of total specification range
Analytical variability Process variability
Reliability of evaluation of major process variables by analytical procedures depends on analytical variability
Impurities
LOQ and specification limit (e.g. qualification limits NMT 0.15%)
Response factors (LOQ modified by response factor)

49. Analytical Method Development
Methods for cleaning validation
Method for assay and related substances used in stability studies of API and FPP
Specificity (in samples taken from a cleaning assessment)
Linearity of response (from 50% of the cleaning limit to 10x this concentration; R2 ≥ )
Precision
Repeatability (RSD ≤ 5%)
intermediate precision [ruggedness (USP)]
Reproducibility
Limits of detection and quantitation
Accuracy or recovery from rinsate (≥ 80%), swabs (≥ 90%), and process surface (≥ 70%)
Range (lowest level is at least 2x higher than LOQ)

50. Analytical Method Development
Summary
Analytical procedures play a critical role in pharmaceutical equivalence and risk assessment/management
Establishment of product-specific acceptance criteria
Assessment of stability of APIs and FPPs
Validation of analytical procedures should demonstrate that they are suitable for their intended use
Validation of analytical procedures deserves special attention during assessment of dossiers for prequalification

51. Analytical Method Development
THANK YOU