Pharmaceutical Development

Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations

Protea Hotel Victoria Junction, Waterfront Cape Town, South Africa Date: 16 to 20 April 2007
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April 2007

Pharmaceutical Development
Analytical Method Development

Presenter:

Jnos Pogny, pharmacist, PhD

pogany.janos@chello.hu

WHO expert

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Analytical Method Development

Outline and Objectives of presentation
Introduction, guidelines Dossier requirements
Assay Related substances Other issues

Main points again

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Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations

Introduction, guidelines

Interchangeability (IC)
INTERCHANGEABILITY (IC) OF MULTISOURCE FPPs = (ESSENTIAL SIMILARITY WITH INNOVATOR FPP) =

PHARMACEUTICAL EQUIVALENCE (PE) + BIOEQUIVALENCE (BE)

IC = PE + BE
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Pharmaceutical equivalence
FPPs meet same or comparable standards (e.g., marketing authorization, analytical methods)
Same API (chemical and physical equivalence) Same dosage form and route of administration Same strength Comparable labeling

Pharmaceutical development equivalence Stability equivalence WHO-GMP (manufacturing equivalence)

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Prequalification requirements
Analytical method validation is required by WHO for the prequalification of product dossiers. Non-compendial ARV APIs and FPPs were/are tested with methods developed by the manufacturer.
Analytical methods should be used within GMP and GLP environments, and must be developed using the protocols and acceptance criteria set out in the ICH guidelines Q2(R1)

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Guidelines used in PQP

WHO-GMP 4.11 It is of critical importance that particular attention is paid to the validation of analytical test methods, automated systems and cleaning procedures.
Appendix 4. Analytical method validation (in WHO Expert
Committee on Specifications for Pharmaceutical Preparations. 40th Report. Geneva, WHO, 2006 (WHO Technical Report Series, No. 937). http://whqlibdoc.who.int/trs/WHO_TRS_937_eng.pdf

Validation of analytical procedures: text and methodology Q2(R1) ICH Harmonized Tripartite Guidelines, (2005)
http://www.ich.org/LOB/media/MEDIA417.pdf

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General requirements
Qualified and calibrated instruments Documented methods Reliable reference standards

Qualified analysts
Sample selection and integrity Change control
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Measure of variation (spread of data)

68.26%

95.46%

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Mean (average) chart

Abnormal variation of process special causes
USL Upper specification limit

average = mean

Normal variation due to common causes

LSL

Lower specification limit

Abnormal variation of process special causes

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Capable process
Almost all the measurements of a stable process fall inside the specification limits
USL LSL Cp 6 8 10 12

1.00 1.33 1.66 2.00 6 ppm 2 ppb

OoS results: .27%.

64 ppm

http://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htm
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NEVIRAPINE Reference Standard

Injection tR A

System suitability requirement: RSD is NMT 0.85%

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1 2 3 4 5 6 Mean STD RSD

6.160 6.167 6.166 6.172 6.165 6.168 6.166 0.004 0.06%

12466 12311 12432 12530 12457 12479 12446 74 0.59%

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Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations

Dossier requirements

Use of analytical methods - generics

Clinical
To determine bioavailability in healthy volunteers

Pharmaceutical
To develop a stable and reproducible formulation for the manufacture of bioequivalence, dissolution, stability and pilot-scale validation batches

Methods
To understand the profile of related substances and to study stability and start measuring the impact of key product and manufacturing process parameters on consistent FPP quality

At initial phase of pharmaceutical development

At advanced phase of pharmaceutical development

To prove bioequivalence after critical variations to the prequalified dossier
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To optimize, scale-up, and transfer a stable and controlled manufacturing process for the prequalification product

To be robust, transferable, accurate, and precise for specification setting, stability assessment, and QC release of prequalified batches

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Analytical procedure characteristics

Type of characteristic Accuracy Precision Specificity Identification + Impurities
Quantitative Limit

Assay + + + +

+ + +

Detection limit Quantitation limit

Linearity Range Robustness
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+
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+
+ + +

+ +

+ + +

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Accuracy - ISO 5725 1-6

Accuracy

Trueness

Precision
(Random errors)

Systematic errors

Intra-assay variability

Intra-laboratory variability

Inter-laboratory variability

Repeatability

Intermediate precison

Reproducibility

Source: ISO. 1994. ISO 5725 1-6: Accuracy (Trueness and Precision) of Measurement Methods and Results. ISO, Geneva, Switzerland.

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Accuracy and precision

Inaccurate and imprecise

Inaccurate & imprecise

Inaccurate but precise

Precise

Accurate
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Accurate but imprecise

Accurate and precise

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Percent accuracy (hypothetical figures)

Sample
1 2 3 4 5 6

LA, %
50 70 80 100 120 130 Mean

Nevirapine, mg Added 0.499 0.703 0.796 1.001 1.211 1.299 0.918 Recovered 0.495 0.701 0.795 1.005 1.209 1.296 0.917

Recovery % 99.2 99.8 99.9 100.4 99.8 99.8 99.8

RSD % 0.64 0.31 0.27 1.88 0.38 1.12 0.77

The data show that the recovery of analyte in spiked samples met the evaluation criterion for accuracy (100 2.0% across 50130% of target concentrations).
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Percent accuracy (hypothetical figures)

104 103 102 101 100 99 98 97 96 0 1 2 3 4 5 6 7 Number of samples

% Recovery

Red line: LA
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Green lines: USL and LSL

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Precision (of any process)

Measured mean Real mean

The precision (VARIABILITY) of an analytical procedure is usually expressed as the standard deviation (S), variance (S2), or coefficient of variation (= relative standard deviation, RSD%.) of a series of measurements. The confidence interval (CI) should be reported for each type of precision investigated.

PRECISION
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Repeatability (of any process)

Measured mean

Repeatability expresses the precision (spread of the data, variability) under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.

REPEATABILITY
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Repeatability (hypothetical figures)

Injection 1 2 Peak area 57935 57833 Imp1 0.301 0.301

3
4 5 6 Mean

57497
57617 57778 57231 57649

0.299
0.300 0.301 0.298 0.300

The repeatability precision obtained by one analyst in one laboratory was 1.25% RSD for the analyte and, therefore, meets the evaluation criterion of RSD 2%.

STD
RSD 95% CI
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257
0.4% 270
April 2007

0.0013
0.4% 0.0014

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Intermediate Precision and Reproducibility (of any process)

Measured means

Intermediate precision expresses within-laboratories variations. #1, #2 and #3: different days, different analysts, different (manufacturing) equipment, etc.

Reproducibility expresses the precision between laboratories #1, #2 and #3 (collaborative studies, usually applied to standardization of methodology). (Transfer of technology)
Intermediate precision or Reproducibility
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Intermediate precision (ruggedness)

Sample 1 2 3 4 5 6 Mean STD RSD 95% CI
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Assay (mg/5ml) 51.7 52.6 51.9 53.0 52.5 52.3 52.7 52.4 0.49 0.9% 0.51
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Combined values Mean STD RSD 95% CI 52.5 0.48 0.9% 0.31

52.1 52.3 52.9 53.2 53.1 52.7 0.44 0.8% 0.46

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Specificity (selectivity)
Specificity is the ability to assess unequivocally the analyte in the presence of components, which may be expected to be present. Typically these might include impurities, degradants and excipients. An example of specificity criterion for an assay method is that the analyte peak will have baseline chromatographic resolution of at least 2.0 minutes from all other sample components
Stability indicating analytical methods should always be specific.
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Identification a special case

Diethylene glycol (DEG) in paediatric dosage forms has been implicated as the causative agent in numerous deaths since 1937. The victims were mainly children. Illustrative analytical issues of investigation
IR identity test was able to detect DEG at about 20 %w/w Testing of DEG in Glycerol (and in Propylene Glycol) was recommended with a LOD (sensitivity) of NLT 0.1 %. For detecting DEG at low levels, GC seemed preferable. The assay was the most relevant test (accurate within 0.2%)

Illustrative regulatory issues

Legislation GMP

Specificity is an essential but not sufficient characteristic of identification

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Specificity (hypothetical figures and data)

HPLC chromatograms of (a) API reference standard, (b) FPP and (c) placebo

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SPECIFICITY degradants
Stress A (%) *

Initial
Acid Peroxide

100.0
99.3 99.8

All others

100.0

Purity angle 0.040 0.105 0.725 0.045 0.120 1.040 0.060 0.110 0.690 NA

Purity threshold 0.280 0.380 1.630 0.280 0.410 1.610 0.270 0.360 1.250 NA

There were no peaks in the placebo chromatogram at the retention times of nevirapine (N), methylparaben (MP) and propylparaben (PP) peaks.

*Sum of N, MP and PP peak areas. The three ingredients can be assessed in the presence of (nonexpected) degradants. The peaks are homogeneous and pure. The method is selective, specific and stability-indicating.
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LOD, LOQ and SNR

Limit of Quantitation (LOQ) Limit of Detection (LOD) Signal to Noise Ratio (SNR)

Peak B LOQ

Peak A LOD
Baseline noise

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LOD and LOQ (hypothetical figures)

Injection
1 2 Impurity 1 LOD 4176 3608 LOQ 7235 8099 Impurity 2 LOD 3497 4258 LOQ 7892 7791

3
4 5 6 Mean STD RSD

4196
4303 3932 5238 4242 548 12.9%

7950
8166 7847 8415 7952 402 5.1%

3275
3464 4008 4702 3867 551 14.3%

8292
8050 8368 8284 8113 238 2.9%

Conc. (g/ml)
Conc. (%w/w)
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0.086
0.017

0.171
0.033

0.107
0.019

0.214
0.039

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LOD and LOQ

The limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be detected, not quantified. It is expressed as a concentration at a specified signal : noise ratio (SNR), usually between 3 and 2 : 1.
In this study, the LOD was determined to be 0.086 g/ml (Impurity 1) with a signal : noise ratio of 3.6 : 1

The limit of quantitation (LOQ) is defined as the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method. The ICH has recommended a signal : noise ratio (SNR) of 10:1.
The LOQ was 0.171 g/ml (Impurity 1) with a signal:noise ratio of 11.3. The RSD for six injections of the LOQ solution was 2%.
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Linearity
Measured mean Real mean

Precision

Linearity expresses differences in precision at different points of a given range. The 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.

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Linearity and range

5000 4000 y = 36.124x - 7.2984 R = 0.9998
2

Assay mean

3000 2000 1000 0 0 20 40 60 80 100 120 140 Concentration, %

Acceptance criterion: correlation coefficient should not be less than 0.9990

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Linearity and range

Concentration range 1.01.3 mg/ml (10130% of the theoretical concentration in the test preparation, n=3) Regression equation was found by plotting the means of peak area (y) against the analyte concentration (x) expressed in %: y = 36.124x - 7.2984 (R2 = 0.9998). The regression coefficient demonstrates an excellent relationship between peak area and concentration of analyte. The analyte response is linear across 10-130% of the target nevirapine concentration.

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Range (minimum requirements)

Assay of an API or a FPP: 20% of the test concentration. Content uniformity: 30% of the test concentration (unless a wider
more appropriate range, based on the nature of the dosage form (e.g., metered dose inhalers), is justified).

Dissolution testing: 20 % over the specified range.

Impurity: from the reporting level of an impurity to 120% of the specification. (Unusually potent or toxic impurities, LOD and LOQ should be
commensurate with ICH requirement.)

If assay and purity are performed together as one test and only a 100% standard is used, linearity should cover the range from the reporting level of the impurities to 120% of the assay specification

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Stability of analytical solution

Stability (of the analytical solution) expresses variation of the measured mean as a function of time.

Stability Measured means

#1 First measurements

#2, #3, #4, n Series of measurements of the same sample within a relatively short period of time.

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Stability of test analytical solution

Time in hours
0 1 2 Impurity-1 Area 72079 71574 71740 0.7% 0.5% Difference

3
4 5 10 15 20 25
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71960
72352 71573 72322 72310 72312 72670
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0.2%
-0.4% 0.7% -0.3%

An analytical solution prepared from Nevirapine 50mg/5ml Oral Suspension was spiked with Impurity-1 at specification level and stored in a capped volumetric flask on a laboratory bench at uncontrolled room temperature under normal lighting conditions for 25 hours.

Conclusion: the stability of the -0.3% analytical solution of Impurity-1 is -0.3% not a source of variation.
-0.8%

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Sensitivity and robustness

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Robustness
Method parameter STP Flow Wavelength Variation of mobile phase Variation -10% 10% -5nm +5nm -2% +2% -5oC +5oC -0.3 +0.3 tR Impurity 1 0.83 0.83 0.84 0.82 0.83 0.80 0.84 0.82 0.83 0.83 0.83 Impurity 2 1.80 1.81 1.82 1.81 1.81 1.89 1.76 1.80 1.81 1.81 1.80

Column temperature
pH
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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)], and 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)
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Main Points Again

Analytical procedures play a critical role in pharmaceutical equivalence and risk assessment / management:
establishment of product-specific acceptance criteria, and stability of APIs and FPPs.

Validation should demonstrate that the analytical procedure is suitable for its intented purpose. HPLC systems and method validation deserves special attention during the assessment of dossiers for prequalification.
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THANK YOU

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