1. VALIDATION PRINCIPLES
Principles of qualification and validation which are
applicable to the manufacture of medicinal products.
Analytical Method אנליזה מעבדתית
Medical Devices אביזרים ומכשור רפואי (אמ"ר)
Cleaning and Sterilization ניקוי ועיקור
Process: Development, Analysis, and Control.
פיתוח ניתוח ובקרת תהליך
Eli. S M.Sc,
Lect. No. 7

2. Objective The objective of validation of an analytical
procedure is to demonstrate that it is suitable for
its intended purpose.
Eli. S M.Sc,
Lect. No. 7

3. Laboratory Method Flow
APPROVED
Method
Development
Method
Validation
Method
Transfer
פיתוח
ואלידציה
העברה
Eli. S M.Sc,
Lect. No. 7

4. Analytical Method Development
Prerequisites for analytical method Validation - Six “M”s
Quality of 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
Eli. S M.Sc,
Lect. No. 7

5. Why Validate
Method validation is completed to ensure that an analytical methodology is accurate, reproducible and rugged over specific range that an analyte will be analyzed.
Provides assurance of reliability אמינות
FDA Compliance התאמה מתועדת לשיטת הבדיקה The process of providing documented evidence that something does what it is indented to do
Eli. S M.Sc,
Lect. No. 7

6. World Health Organization
Validation
World Health Organization
27 January, 2018
To discuss various aspects of analytical method validation including:
Principles of analytical method validation
Pharmacopoeia methods
Non-pharmacopoeia methods
Approaches to analytical method validation
Characteristics of analytical procedures
Eli. S M.Sc,
Lect. No. 7

7. Validation Principles
World Health Organization
27 January, 2018
Guideline presents information on the characteristics to be considered
Manufacturers to demonstrate - analytical procedure is suitable for its intended purpose (validation)
Validate analytical methods - whether they indicate stability or not
Validated by R&D before being transferred to the quality control unit when appropriate
1. Principle
1.1 This appendix presents some information on the characteristics that
should be considered during validation of analytical methods. Approaches
other than those specifi ed in this appendix may be followed and may be
acceptable. Manufacturers should choose the validation protocol and procedures
most suitable for testing of their product.
1.2 The manufacturer should demonstrate (through validation) that the
analytical procedure is suitable for its intended purpose.
1.3 Analytical methods, whether or not they indicate stability, should be
validated.
1.4 The analytical method should be validated by research and development
before being transferred to the quality control unit when appropriate.
Eli. S M.Sc,
Lect. No. 7

8. Validation Principles (Cont’)
World Health Organization
Validation Principles (Cont’)
27 January, 2018
Specifications for materials and products, with standard test methods
Manufacturer to use “pharmacopoeial specifications and methods”, or suitably developed “non-pharmacopoeial specifications and methods” as approved by the national drug regulatory authority
Use well-characterized reference materials, with documented purity, in the validation study
2. General
2.1 There should be specifi cations for both, materials and products. The
tests to be performed should be described in the documentation on standard
test methods.
2.2 Specifi cations and standard test methods in pharmacopoeias (“pharmacopoeial
methods”), or suitably developed specifi cations or test methods
(“non-pharmacopoeial methods”) as approved by the national drug regulatory
authority may be used.
2.3 Well-characterized reference materials, with documented purity,
should be used in the validation study.
Eli. S M.Sc,
Lect. No. 7

9. World Health Organization
Validation
World Health Organization
27 January, 2018
Pharmacopoeial / Non-pharmacopoeial methods
Pharmacopoeial methods:
prove that the methods are suitable for routine use in the laboratory (verification)
for determination of content or impurities in products, demonstrate that method is specific for the substance under consideration (no placebo interference)
Non-pharmacopoeial methods:
Should be appropriately validated
3. Pharmacopoeial methods
3.1 When pharmacopoeial methods are used, evidence should be available
to prove that such methods are suitable for routine use in the laboratory
(verifi cation).
3.2 Pharmacopoeial methods used for determination of content or impurities
in pharmaceutical products should also have been demonstrated to be specifi c
with respect to the substance under consideration (no placebo interference).
4. Non-pharmacopoeial methods
4.1 Non-pharmacopoeial methods should be appropriately validated.
Eli. S M.Sc,
Lect. No. 7

10. World Health Organization
Validation
27 January, 2018
Tests include:
Identification tests
Assay of drug substances and pharmaceutical products
Content of impurities and limit tests for impurities
Dissolution testing and determination of particle size
Results should be reliable, accurate and reproducible
2.4 The most common analytical procedures include identifi cation tests,
assay of drug substances and pharmaceutical products, quantitative tests for
content of impurities and limit tests for impurities. Other analytical procedures
include dissolution testing and determination of particle size.
2.5 The results of analytical procedures should be reliable, accurate and
reproducible. The characteristics that should be considered during validation
of analytical methods are discussed in paragraph 6.
Eli. S M.Sc,
Lect. No. 7

11. World Health Organization
Validation
27 January, 2018
When should verification or revalidation be done ?
changes in the process for synthesis of the drug substance
changes in the composition of the finished product
changes in the analytical procedure
transfer of methods from one laboratory to another
changes in major pieces of equipment instruments
Validation extent depends on the nature of the change(s)
Evidence of “analyst proficiency”
2.6 Verifi cation or revalidation should be performed when relevant, for
example, when there are changes in the process for synthesis of the drug substance;
changes in the composition of the fi nished product; changes in the
analytical procedure; when analytical methods are transferred from one laboratory
to another; or when major pieces of equipment instruments change.
2.7 The verifi cation or degree of revalidation depend on the nature of the
change(s).
2.8 There should be evidence that the analysts, who are responsible for
certain tests, are appropriately qualifi ed to perform those analyses (“analyst
profi ciency”).
Eli. S M.Sc,
Lect. No. 7

12. USP – Analytical Method Validation (In Details)
דיק - PRECISION
דיוק - ACCURACY
LIMIT OF DETECTION (LOD)
Method
Validation
LIMIT OF QUANTIFICATION (LOQ)
ייחודי - SPECIFICITY
LINEARITY AND RANGE
"קשיחות" - RUGGEDNESS
Eli. S M.Sc,
Lect. No. 7
ROBUSTNESS "חוסן" -

13. Precision Definition דיק -
The closeness of agreement between the values obtained in an assay. When the method is applied repeatedly to multiple sampling of a homogenous sample
Expressed as % SD for a statistically significant number of samples
Should be performed at 3 levels.
Repeatability (הדירות)
Intermediate Precision (דיק ביניים)
Reproducibility (שעתוק)
Eli. S M.Sc,
Lect. No. 7

14. Precisionדיק - Explanation and Enlargement
Repeatability: -הדירות (USP analytical Procedure Criterion) Same operating condition, short time interval. Inter-assay Precision Within-lab variations: Different days, analysts, equipment. Multiple determinations of a single sample, controls and reagents analyzed in several runs, same lab.
Eli. S M.Sc,
Lect. No. 7

15. Precisionדיק - Explanation and Enlargement
Minimum of 9 determinations covering specified range of procedure (3 levels 3 replica. each), or Minimum of 6 determination at 100% test conc. (expressed as % CV) Reproducibility ( (שעתוק Precision between labs. Collaborative studies, (not relevant in manufacturing facility)
Eli. S M.Sc,
Lect. No. 7

16. Accuracy Definitionדיוק -
The closeness of agreement between the actual values (ערך אמיתי)of the drug and the measured value (ערך נמדד)
Spiked (known added amount) and
Recovery studies are performed to
measure accuracy
Eli. S M.Sc,
Lect. No. 7

17. Accuracy Definitionדיוק – (Cont’)
Expressed as % error between the observed value and the true value
(Observed value – True Value)x100
True value
Good accuracy means minimum Bias
Eli. S M.Sc,
Lect. No. 7

18. Accuracy vs Precision Accuracy Precision
Closeness of agreement between true value and the mean of observed value obtained
over large number of observations. This can be quantitatively expressed as
Bias (סטיה/נטיה)
Observed value – True value
Good accuracy means minimum Bias.
Precision
Precision is closeness of results with each other of a large number of successive
observations in a measurement process, under prescribed conditions. This can be
quantitatively expressed as percentage of coefficient of variation ( % CV).
Good precision means minimum % CV. It can be calculated using a formula
Precision can be demonstrated through repeatability and reproducibility.
Eli. S M.Sc,
Lect. No. 7

19. Repeatability vs Reproducibility
(a) Repeatability : Closeness of the agreement between the successive
measurements of the same specimen and with the following conditions:
§ The same measurement procedure
§ The same analyst
§ The same measurement systems used under the same conditions
§ The same location
(b) Reproducibility : Closeness of the agreement between the results of
successive measurements of the same specimen and with the following conditions:
§ Different analysts
§ Different measuring systems
§ Different locations and at different times
Eli. S M.Sc,
Lect. No. 7

20. Accuracy Determination קביעת דיוק
(Cont’)
Drug Substance
Analysis of Reference material Compare results to a second, well characterized method
Impurities (Quantification)
Analysis of Samples (Drug substances) spiked, with known amounts of impurities
Eli. S M.Sc,
Lect. No. 7

21. Analytical Method Development
Accuracy vs. Precision
Accurate & Precise
Accurate & imprecise
Inaccurate &
precise
Inaccurate & imprecise
Eli. S M.Sc,
Lect. No. 7

22. Limit of Detection (LOD) Definition
גבול זיהוי
Lowest Concentration of Analyte in a Sample that can be detected (not necessarily quantitated)
Limit Test: Above or Below a Certain Level – Pass / Fail results
Expressed as Concentration (%, ppb, mg/ml)
Eli. S M.Sc,
Lect. No. 7

23. Limit of Detection (LOD) Definition (Cont’)
Almost never necessary to be determined
May be based on standard deviation (STD) of the response and slope
DL=(3.3) STD / S
STD = Standard Deviation of the response
S = Slope of calibration curve
Eli. S M.Sc,
Lect. No. 7

24. Limit of Quantification (LOQ) Definition
Lowest concentration of Analyte in a sample that can be determined with acceptable precision and accuracy under stated operational conditions
Expressed in Concentration of Analyte
Signal To Noise Ratio (10:1 is typical)
Eli. S M.Sc,
Lect. No. 7

25. Limit of Quantification (LOQ) Explanation and Enlargement
QL = (10) STD / S
STD = Standard Deviation of the response
S = Slope of Calibration curve
DL and QL are validated by analyzing a
suitable number of samples
Method should be documented !!!
Eli. S M.Sc,
Lect. No. 7

26. World Health Organization
27 January, 2018
LOQ, LOD and SNR
Limit of Quantification
Limit of Detection
Signal to Noise Ratio
Peak B LOQ
There are no specific criteria set for the Limit of Quantitation (LOQ) and Limit of Detection (LOD) but guidance is available from specifications and pharmacopeias.
The noise is measured by running the instrument at maximum gain with no test being processed. The ripple generated is noise due to the instrument’s electronics, etc. The peak is measured relative to this noise and the ratio is calculated. This is known as the Signal to Noise Ratio (SNR).
Generally:
LOD SNR should be greater than 2:1. Peak A is acceptable for LOD but not for quantitation;
The LOD can be calculated if the standard deviation (SD) of the response (which is standard deviation of the blank) and the slope is determined:
LOD = 3.3 x SD
slope
Similarly LOQ = 10 x SD
The LOQ SNR should generally be above 10:1. Peak B is suitable for quantitation;
Precision as a percentage relative standard deviation should be % at the limit for LOQ.
Peak A LOD
noise
Baseline
Eli. S M.Sc,
Lect. No. 7

27. Specificity (Selectivity) Definition
The ability to measure accurately and specifically the Analyte in the presence of components that may be expected to be present in the product
The Degree of Interference
Active Ingredients
Excipients
Impurities
Degradation Products
Placebo Ingredients
Eli. S M.Sc,
Lect. No. 7

28. Specificity Determination
Qualitative Identification Test
Demonstrate ability to select between compounds of closely related structure
Confirm Positive and Negative results
Assay
Demonstrate that the results are unaffected by spiked impurities or excipients
Eli. S M.Sc,
Lect. No. 7

29. Linearity and Range ליניאריות ותחום
The ability of the method to elicit test results that are directly proportional to concentration within a given range
- Expressed as the Variance of the Slope of the Regression Line
Range
Interval between upper and lower levels of Analyte demonstrated by the method
Eli. S M.Sc,
Lect. No. 7

30. World Health Organization
Linearity and Range
27 January, 2018
Table of values (x,y) x y  #
Reference material mg/ml
Calculated mg/ml 1
0.0100
0.0101 2
0.0150
0.0145 3
0.0200
0.0210 4
0.0250
0.0260 5
0.0300
0.0294 6
0.0400
0.0410
This is a typical plot of raw data to check for linearity and range.
The table of raw data on the right hand side has been plotted using linear regression to obtain a line of best fit. The x axis is the reference material that was weighed out and the y axis the calculated values from the instrument readings. These are plotted (the blue line with the points as a yellow box and error bars) and a linear regression analysis is performed.
The two yellow lines are the 2-sided 95% confidence limits.
Eli. S M.Sc,
Lect. No. 7

31. Prepare Minimum 5 Concentrations
Linearity Evaluation
Prepare Minimum 5 Concentrations
(Preferable 6-8 sample dilutions across the range)
Test each dilution in triplicates
for 3 runs. (5x3x3=45)
Eli. S M.Sc,
Lect. No. 7

32. Linearity Evaluation Record expected values, actual values,
and % recovery for each one.
Analyze each set of dilutions for statistical methods: Regression, Correlation Coefficient, y-intercept, Slop, Plot.
Eli. S M.Sc,
Lect. No. 7

33. Ruggedness "קשיחות"or Robustness "חוסן"
Degree of Reproducibility of Test Results
under a Variety of Conditions
Different: Laboratories, Analysts, Instruments, Reagents, Days
Expressed as % RSD
Eli. S M.Sc,
Lect. No. 7

34. Ruggedness "קשיחות"or Robustness "חוסן"
Measure The Capacity to Remain Unaffected by Small Variation in Method Parameters.
Indication or Reliability During Normal Use.
Eli. S M.Sc,
Lect. No. 7

35. Why is Method Validation Considered a Bottleneck צוואר בקבוק -
Every Method validation Process Consists About Analyses
Each of Them Produce About 4 specific Results; These results in a total of about Numbers
Eli. S M.Sc,
Lect. No. 7

36. Why is Method Validation Considered a Bottleneck (Cont’)
All of These Numbers Need to be mathematically Processed:
Manual (Calculator, Excel Macro)
Dedicated Method Validation Program
Eli. S M.Sc,
Lect. No. 7

37. Evaluation of analytical validation data Data provided by applicant
The objective of the analytical procedure
The analytical technique
Item
Data provided by applicant
(very briefly)
Acceptable or not? (add comments if necessary, & reasons if unacceptable)
Is a chromatogram, spectrum or similar provided?
Specificity
Linearity
Range
Accuracy
Precision: Repeatability
Precision: Intermediate
Precision: Reproducibility
Detection limit
Quantitation limit
Robustness
System suitability (if necessary)
Data on the reference standard
Other evaluator comments:
Lect. No. 7
Eli. S M.Sc,

38. - Cost / Benefit Economic Exercise
Why Revalidate
To Take Advantage of a New Technology
- Cost / Benefit Economic Exercise
- Instrumentation, Methods
Formulation Changes
Eli. S M.Sc,
Lect. No. 7

39. Why Revalidate (cont’)
Manufacturing Batch Changes
Changes in Incoming Raw Material
Changes in Method
Eli. S M.Sc,
Lect. No. 7

40. CONCLUSION סיכום Defined Validation and its Importance
Examined The steps of Method Validation
Learned How each Step is Documented and
Measured: (System Suitability, USP/ICH)
Protocol and Pitfalls: Revalidation
Eli. S M.Sc,
Lect. No. 7

41. Kinetic LAL System Principles
Btoforum 2007
Kinetic LAL System Principles
O.D. Change Over Time (Onset OD)
Bioforum 2007
E. Solomon M.Sc,
Eli Solomon M.Sc, כל הזכויות שמורות

42. Kinetic LAL System Principles
Btoforum 2007
R > 0.98
R > 0.98
Bioforum 2007
E. Solomon M.Sc,
Eli Solomon M.Sc, כל הזכויות שמורות

43. Bioforum 2007
E. Solomon M.Sc,

44. Bioforum 2007
E. Solomon M.Sc,

45. ANALYTICAL METHOD VALIDATION REFERENCE
Initial Method Validation Guidance -1987
Guidance for submitting samples and analytical data for methods validation. FDA, February US
Government Printing office: :20818.
Analytical Procedures and Method Validation Fed.Reg ), 52,776-52,777, 30 August 2000
All Guidance are on FDA Web site:
Eli. S M.Sc,
Lect. No. 7

46. ANALYTICAL METHOD VALIDATION REFERENCE
Pharmaceutical Process Validation, edited by: R. A. Nash; Alfred H. Wachter, An International Third Edition, Revised and Expanded, Marcel Dekker, Inc. New York, Basel, Hong Kong (2003).
Marjo-Riitta Helle et al.: A literature review of pharmaceutical process validation, 52 references, Pharmaceutical Technology Europe, August 2003.
Eli. S M.Sc,
Lect. No. 7