1. Course: BioT 006 Quality Control & Validation
Assay Validation
Course: BioT 006
Quality Control & Validation

2. Definitions: (Dictionary: Relative to quality endeavors)
Validation: An act, process, or instance to
support or corroborate something
on a sound authoritative basis.
Verification: An act or process of establishing
the truth or reality of something.
Qualification: An act or process to assure
something complies with some
condition, standard, or specific
requirements.

3. Why Validate? Quality by design, built in Can’t inspect quality in
Demonstrate control of process
Good science
Good business
It’s the law

4. Considerations Prior to Validation
Suitability of Instrument
Status of Qualification and Calibration
Suitability of Materials
Status of Reference Standards, Reagents, Placebo Lots
Suitability of Analyst
Status of Training and Qualification Records
Suitability of Documentation
Written analytical procedure and proper approved protocol with pre-established acceptance criteria

5. Analytical Methods Validation
Considered a critical step in the manufacturing process
Requirements for validated analytical methods explicitly written into the CFR’s
– Testing and release for distribution
FDA: Requires that the:
Accuracy
Sensitivity
Specificity
Reproducibility of test methods etc.
Employed by the firm shall be established and documented.
Such validation and documentation:
Accomplished in accordance with 21 CFR (a)(2)

6. What is not an Analytical Method Validation?
Calibration
The Process of Performing Tests on Individual System/ Components to Ensure Proper function
For example:
HPLC Detector calibration, pH meter
Analytical Balance, Centrifuge
Particle counter etc.

7. Compendium Compendium: Compendial:
A collection of concise but detailed information about a particular subject, especially in a book or other publication.
Compendial:
Pertaining to compendium

8. Verification vs. Validation
Compendial vs. Non-compendial Method
Compendial methods: Verification
Non-compendial methods: Validation requirement

9. Validation protocol for analytical method
Statement of purpose and scope
Responsibilities
Documented test method
List of materials and equipment
Procedure for the experiments for each parameter
Statistical analysis
Acceptance criteria for each performance parameter

10. Validation Component Specificity (Selectivity) Accuracy Precision
Linearity
Detection limit
Quantitation Limit
Range
Robustness
Ruggedness

11. Specificity
The ability of the analytical method to quantify (differentiate) the analyte in the presence of other components in the sample
Does the analytical method detect the component it is supposed to detect?
e.g. Cross reactivity in antibody based methods
Demonstrate specificity by conducting analytical method on materials that may mimic analyte of interest
Looking for “false positives”

12. Specificity, contd. Specificity may be affected by: Interference:
Excipients or impurities
Spike effect:
Adding appropriate levels
Assay results should be unaffected
Chromatography or Electrophoresis
Impurities get separated

13. Accuracy/ Trueness
Ability of analytical method to accurately determine the presence and amount of the analyte of interest
The closeness of mean test results to the true value of the analyte.
It is determined by replicate analysis of samples containing known amounts of the analyte.
Accuracy should be measured using a minimum of five determinations per concentration.
A minimum of three concentrations in the range of expected concentrations is recommended.

14. Accuracy
Accuracy is hitting the part of the bulls eye for which you’re aiming at.

15. Precision
The closeness of agreement (degree of scatter):
Between a series of measurements obtained from multiple sampling of the same homogeneous sample
How much variability does the assay exhibit when analyzing the same sample
Precision should be measured using a minimum of five determinations per concentration.
A minimum of three concentrations in the range of expected concentrations
Measured by parameters of variation, mostly CV (Coefficient of variation)

16. Coefficient of variation
Also called Relative Standard Deviation
CV = Coefficient of variance
It is a measure of dispersion of a probable distribution
RSD = SD (or Sigma) X 100
Mean
= CV

17. Precision… Considered at 2 Levels
Repeatability (Intra assay)
Intermediate Precision (Inter assay)

18. Precision: Intra-assay (Repeatability)
Express the precision under the same operating conditions over a short interval of time.
Should be assessed using minimum of 15 determinations
5 concentrations/ 3 replicates
Minimum of 5 determinations within the range.

19. Precision: Inter-assay
Express variations within-laboratory.
Expressed in terms of relative standard deviation
coefficient of variation
Studies should include varying days, analysts, equipment, etc.
Also called Intermediate precision

20. Linearity & Range
Linearity: The ability of the assay (within a given range) to obtain test results which are:
Directly proportional to the concentration/amount of the analyte
Range: The interval between the upper & lower concentrations of an analyte for which:
the assay has suitable levels of
Precision
Accuracy
Linearity

21. Linearity & Range (cont.)
Linearity and Range can be evaluated simultaneously
Range is established by confirming acceptable degrees of:
Linearity
Accuracy
Precision
within or at the extremes of a specified range

22. Linearity Should be Evaluated
By Visual Inspection of plot of signals vs. analyte concentration
By Appropriate statistical methods
Linear Regression (y = mx + b)
Requires a minimum of 5 concentration levels

23. Method Validation LOD & LOQ III
Lowest level at which method can detect analyte (LOD or DL)
Lowest level at which method can accurately quantify analyte (LOQ or QL)
Precision and LOQ related
Lower LOQ will typically result in lower precision

24. Method Validation LOD & LOQ IV
A method is not acceptable for accurate
detection or quantitation if:
the analyte level is likely be fall beneath the limit(s) calculated based upon the blank signal and its standard deviation
Mean sample signal must be sufficiently larger than the blank
so that positive detection or accurate quantitation is possible

25. Ruggedness
The effectiveness of an analytical process in face of small environmental/operating conditions, such as:
Different analysts
Different equipment
Different labs
Different time

26. Method Validation Ruggedness
Methods need to be shown to be rugged
A replicate set of data produced at a particular time point by an operator working with a particular set of equipment in a given laboratory will verify repeatability.
To show reproducibility, the method must produce similar results when any of these parameters are changed.
The most likely changes are to time and operator.

27. Validation: Robustness
Robustness of an analytical method refers to:
It’s ability to remain unaffected when subjected to small changes in method parameters.
e.g. in PCR: Defining the critical range of:
Primer concentration
Mg2 Concentration
Thermo cycler temperature range
e.g. in HPLC:
A perfect mobile phase is one which allows small changes in the composition without affecting the selectivity or the
quantitation of the method.
Usually a part of the assay optimization
Prior to the validation process

28. Characteristics of Analytical Procedures
Ruggedness:
It is due to factors external to the method
Robustness
It is due to factors internal to the method.
Variability caused by:
Day-to-day variations
Analyst-to-analyst
Laboratory-to-laboratory
Instrument-to-instrument
Chromatographic column-to-column
Instability of analytical reagents
Reagent kit-to-kit

29. Validation: Method Performance
Characteristics
Purity
Impurity Detection
Impurity Quantitation
Identity
Content
Accuracy +
Specificity
Precision Repeability
Precision Intermediate
Linearity
Range
LOQ
LOD

30. Extent of Validation New methods require complete validation
Pharmacopoeia methods require partial validation (or verification)
Significant changes mean partial revalidation
equipment changes
formula changed
changed suppliers of critical reagents

31. Bradford Assay for Total Protein
Well known colorimetric assay that relies on:
the binding of Commassie Blue G-250 dye to the proteins in an acidic solution
Dye binding is proportional to number of positive charges in protein
Color (absorbance) is read at 595 nm
Proteins up to 3000 Da not detected
Assay linear over a short range, typically from 0 µg/mL to 2000 µg/mL,
often making dilutions of a sample necessary before analysis.
Simple, quick, wide range, few interfering agents

32. Bradford Assay Accuracy Specificity Precision Linearity & Range
Limit of detection (LOD)
Limit of quantification (LOQ)
Robustness

33. Validation: Analytical method transfer
Method transfer protocol and procedure
Example: R&D lab to QC Lab
Precision
Accuracy
Ruggedness
Written and approved specific test method
Proficiency check
Three assays compared to original lab assay
Formal acceptance by new laboratory

34. Chemical Laboratory Validation Requirements
Balances
Chromatography
HPLC, GC, TLC
Dissolution or disintegration apparatus
Karl Fischer moisture determination
Melting, softening or freezing point apparatus
Ovens, refrigerators, incubators
pH meter
Spectrophotometer UV/Vis, IR, Amino Acid Analyzer
Timers
Viscometer
Volumetric equipment/glassware

35. Typical validation of HPLC assay
Method validation
Specificity: that the method is free of interference from excipients, impurities, etc.
Accuracy: that the method gives closeness to true results.
Precision: by checking that the method is precise.
Linearity: that the method will produce results that are directly proportional to the concentration of analyte in the samples.
Robustness: by checking that the method will withstand deliberate changes.

36. Microbiological Testing: Validation
Microbial limit testing:
indicator organisms such as S. aureus, P. aeruginosa etc.
Microbial count:
Total viable count in starting and finished products
Sterility testing
Preservative effectiveness testing:
After inoculation, bacteria should not grow in up to 28 days
Environmental monitoring program:
Water air and surface monitoring

37. Validation: Microbial test procedures
Incubation temperature and time
Media may not grow all microorganisms
Variations in media may affect recovery: shelf life
Inhibitory disinfectants or preservatives: water with chlorine must be neutralized
Sample
procedures
handling, storage, transport
In case the sample is contaminated when the sample is drawn, or very long storage and very hot or cold storage temperatures are used, then the sample will be affected.

38. Sterility Testing Validation
Media growth promotion, sterility, pH
Product validation
Stasis testing: Also called inhibition test, 
It is performed to ensure that there are no inhibitory substances remaining in the product and  that the media is still capable of supporting the growth of microorganisms at the end of the sterility test incubation period. 
Environmental monitoring
Negative controls
Challenge organisms:
Staphylococcus aureus, Bacillus subtilis, Pseudomonas aerugihosa, Aspergillus niger, Clostridium sporogenes, and Candida albicans.
For each challenge organism, the inoculum level is CFU.
Validation tests using live microorganisms must not be undertaken in the sterility testing area.

39. Re-Validation When What Method parameters have been changed
The scope of the method has been changed
Synthetic methods have been changed
Impurity profile has been changed
What
Preferably everything
Exceptions should be scientifically justified