Analy Meth Dev 06.12.09

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
1| Birgit Schmauser | April 2008

Analytical Method Development
Presented by:
Birgit Schmauser, PhD

Federal Institute for Drugs
and Medical Devices (BfArM)
b.schmauser@bfarm.de

In this presentation:
 Standards in developing analytical methods for

– Originator and multisource generic FPPs
• Specifications
• Stability
 Parallel development of analytical methods for

cleaning validation

 Originator, First-time Generic and Multisource Generic

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

 HPLC-method to assay potency and purity – risk assessment

Originator First-time Generic Multisource Generic
Selectively screen/detect any
impurity or degradant
Establish potency
Identify impurities/degradants
Characterise all Derive impurities/degradants from Verify impurities from

impurities/degradants Calculate Originator Pharmacopoeia
Response factors Characterize „in-house“ Characterise „in-house“
(qualification by clinical use) impurities/degradants impurities/degradants
Calculate response factors (Response factors)
Establish reference materials Extract (& reproduce) reference Use pharmacopoeial reference
materials materials
Adapt to routine use Adapt/modify to/for routine use „Implement“ for routine use

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

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)

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

Use of analytical methods - generics
CLINICAL PHARMACEUTICAL METHODS
At initial phase of pharmaceutical development
To determine To develop a stable and To understand the profile of related
bioavailability in healthy reproducible formulation for the substances and to study stability
volunteers manufacture of bioequivalence, To start measuring the impact of key
dissolution, stability and pilot-scale product and manufacturing process
validation batches parameters on consistent FPP quality
At advanced phase of pharmaceutical development

To prove bioequivalence To optimise, scale-up and transfer To be robust, transferable, accurate
after critical variations to a stable and controlled and precise for specification setting,
the prequalified dossier manufacturing process for the stability assessment and QC release of
prequalification product prequalified product batches

Prerequisites for analytical method validation
– Six “M”s
Man Machine Methods
qualified calibrated characterised
robust documented
skilled qualified suitable
Quality of the
Reference analytical method
Vibrations Time
standards Irradi- Analysts´
ations support
Tempe-
Quality rature Humidity Supplies
Material Milieu Management
10 | Birgit Schmauser | April 2008

Method development life cycle
Planning Method development
Development and Validation Policy Initital Method Development
Development
Pre-Validation Evaluation
Objectives/Requirements of Method Plan –
Project Method Optimization
Information Gathering
Robustness
Resource Gathering System Suitability
Customer Evaluation
Validation Experiments
Testing
Periodically
Method Transfer Monitoring/Review
Filed Method in Use of Methods
Experiments
in Control Labs
. From: Analytical Chemistry in a GMP Environment. Edited by J.M. Miller and J.B. Crowther, ISBN 0-471-31431-5, Wiley & Sons Inc

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

 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

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

 Validation Characteristics
Identification Impurities Assay
quantitative limit
Accuracy - + - +
Precision - + - +
Specificity + + + +
Detection Limit - - + -
Quantitation Limit - + - -
Linearity - + - +
Range - + - +
Robustness + + + +

Accuracy and precision
Accurate &
precise Accurate &
imprecise
&Inaccurate
precise Inaccurate & imprecise

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)

 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 = nσ is described by the “erf-function” ( µ = mean):
xn P
Number of times each value occurs

σ 0.6826895
2σ 0.9544997
3σ 0.9973002
4σ 0.9999366
 An interval of ± 3 σ
5σ99.73%
covers 0.9999994
of values
2σ σ Valuesσ
2σ
3σ 3σ

 Normal distribution, probability function [P(x)] and confidence
interval [CI]
– Probability-P Confidence interval [CI]
centered around the mean [µ]
P xp
in units of sigma [σ ] described by
“inverse erf-function”: 0.800 1.28155σ
0.900 1.64485σ
0.950 1.95996σ
– A CI of 95% includes values
0.995 2.57583σ
± 1.95 σ around the mean
0.999 3.29053σ

 Relationship of variability, probability and reliability of data
– High variability of data (large σ ) generate large confidence intervals and
thus lower the reliability of the mean
– Low variability of data (small σ ) generate small confidence intervals and
thus increase the reliability of the mean

 Repeatability
– Six replicate sample preparation steps from a homogenously prepared tablet
mixture (nominal value of API 150 mg)
Injection Peak area Assay
1 173865 147.10 mg/98.06%
2 174926 148.00 mg/98.66% Mean ± 3 SD =
3 172933 146.32 mg/97.54% Confidence interval of 99.73%
4 175011 148.08 mg/98.72%
5 179557 151.95 mg/101.30% 3x1.32% = 95% - 102.92%± 98.96
6 176425 149.28 mg/99.52%
Mean 175453 148.45 mg/98.96%
SD (σ ) 2329 1.98 mg/1.32%
RSD 1.32% 1.32%

 Intermediate precision
– Expresses within-laboratories variations (different days, different analysts,
different equipment etc.)
Injection Peak area Peak area Peak area

analyst 1 analyst 2 analyst 3
)Mean ± 3 SD: (177252 ≅ 100%
1 173865 175656 177965
2 174926 175878 178556 Analyst 1: 98.96% ± 3 x 1.32%
3 172933 176004 177342 Analyst 2: 99.12% ± 3 x 0.28
4 175011 176344 178011 Analyst 3: 100.70% ± 3 x 0.51
5 179557 175332 179466
6 176425 174959 179688 Average of 3 analysts ± 3SD:
Mean 175453 175695 178504
SD (σ ) 2329 495 918 95% - 102.23%
RSD 1.32% 0.28% 0.51%

 Reproducibility
– Expresses the precision between laboratories
• Collaborative studies, usually applied to

standardisation of methodology
– Transfer of technology
– Compendial methods

 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“
mean true

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

 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 0-471-25953-5, Wiley
& Sons

 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

 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

 Specificity: Overlay chromatogram of an impurity solution with a
sample solution

& Sons

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

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

 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 (σ ) and Slope (S)
• 3.3 σ /S

 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 (σ ) and Slope (S)
• 10 σ /S

) LOD, LOQ and Signal to Noise Ratio (SNR
LOQ
Signal to Noise = 10:1
LOD
Signal to Noise = 3:1
Noise

 LOQ
– Quantitation by SNR is accepted
– Quantitation by Standard deviation of response (σ ) and Slope (S)
(10 σ /S) is more adequate as it involves the response of the
actual analyte
– Best to calculate in the region close to y-intercept

 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

 Spiking
– API test concentration (normalised)
• 0.1 mg/ml (100%)
– Impurity spiking concentrations
• 0.001 mg/ml (1%) – specification limit
• 0.0001 mg/ml (0.1%) – limit of quantitation
(minimum requirement)
API at test concentrations
API below test concentrations

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

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 0-471-25953-5, Wiley & Sons

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

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

 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

& Sons

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

 Influence of buffer pH and buffer concentration in mobile phase
on retention times of API and impurities
API Impurity A Impurity B Impurity C
As is 10.46 3.86 7.43 8.26
buffer pH 5.9 10.45 3.94 7.51 8.38
buffer pH 6.9 10.46 3.94 7.49 8.34
Buffer conc. 83% 7.84 3.43 6.16 6.66
Buffer conc. 87% 15.26 4.77 9.61 11.18
 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

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

 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

When to be „surprised“ about validation data:
– Precision of System precision % RSD 0.33 – 2.25

impurity determination Method precision % RSD 0.0
– Precision of Average peak area % RSD 0.08

API determination Acceptance criterion % RSD ≤ 2.0
– Method precision of Average peak area % RSD 0.4

released API (dissolution) Acceptance criterion % RSD ≤ 10.0

Specification range (USL-LSL)
– Process variability (usually ± 2 SD)
– Analytical variability (± 3σ )
• ~ 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)

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 ≥ 0.9900)
• 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)

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

THANK YOU

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Pharmaceutical Development with Focus

WHO/FIP Training Workshop

Sahar Airport Road

Andheri East, Mumbai, India

28 April 2008 – 2 May 2008

1| Birgit Schmauser | April 2008

Birgit Schmauser, PhD

2| Birgit Schmauser | April 2008

 Standards in developing analytical methods for

 Parallel development of analytical methods for

3| Birgit Schmauser | April 2008

 Originator, First-time Generic and Multisource Generic

4| Birgit Schmauser | April 2008

 HPLC-method to assay potency and purity – risk assessment

Characterise all Derive impurities/degradants from Verify impurities from

5| Birgit Schmauser | April 2008

6| Birgit Schmauser | April 2008

7| Birgit Schmauser | April 2008

8| Birgit Schmauser | April 2008

At advanced phase of pharmaceutical development

9| Birgit Schmauser | April 2008

Material Milieu Management

10 | Birgit Schmauser | April 2008

11 | Birgit Schmauser | April 2008

Validation should verify the suitability of an

12 | Birgit Schmauser | April 2008

 The validation process cannot proceed until the protocol and

13 | Birgit Schmauser | April 2008

14 | Birgit Schmauser | April 2008

15 | Birgit Schmauser | April 2008

16 | Birgit Schmauser | April 2008

17 | Birgit Schmauser | April 2008

Number of times each value occurs

18 | Birgit Schmauser | April 2008

19 | Birgit Schmauser | April 2008

20 | Birgit Schmauser | April 2008

21 | Birgit Schmauser | April 2008

Injection Peak area Peak area Peak area

22 | Birgit Schmauser | April 2008

• Collaborative studies, usually applied to

23 | Birgit Schmauser | April 2008

24 | Birgit Schmauser | April 2008

 Drug product (assay)

 Drug substance/Drug product (Impurities)

25 | Birgit Schmauser | April 2008

From: Analytical Method Validation and Instrument Performance Verification, Edited by

26 | Birgit Schmauser | April 2008

27 | Birgit Schmauser | April 2008

28 | Birgit Schmauser | April 2008

From: Analytical Method Validation and Instrument Performance Verification, Edited by

29 | Birgit Schmauser | April 2008

Assure that known impurities/degradants can be specifically assessed in the

By peak purity assessment and (overlay of) chromatograms

30 | Birgit Schmauser | April 2008

31 | Birgit Schmauser | April 2008

 Determination is usually based on

32 | Birgit Schmauser | April 2008

 Determination is usually based on

33 | Birgit Schmauser | April 2008

) LOD, LOQ and Signal to Noise Ratio (SNR

Signal to Noise = 10:1

34 | Birgit Schmauser | April 2008

35 | Birgit Schmauser | April 2008