Method validation

Question 1

Analytical Method Validation Considerations :-
When we validate :-

Answer 1

Development of new methodology for manufacturing support
Continuous Improvement
To support changes to analytical methodology
Manage obsolescence changes
To show equivalence with pharmacopoeia methods
Raise Change Control
Follow principles outlined in ICHQ2
Purpose of validation is to show that a method is accurate, precise & specific.

Question 2

Accuracy is

Answer 2

the degree to which the result of a measurement, calculation, or specification conforms to the correct value or a standard

Question 3

Precision is

Answer 3

the closeness of two or more measurements to each other is known as the precision of a substance.

Question 4

Specificity is

Answer 4

Defined as the ability of an assay to distinguish target from nontarget analytes, including matrix (i.e., specimen) components.

Question 5

Limit of detection is

Answer 5

The minimum value that can be reproducibly detectable in an analytical method

Question 6

Limit of quantification is

Answer 6

The minimum level of an analyte that can be safely and reproducibly reported

Question 7

Linearity is

Answer 7

The capability of a method to accurately and precisely determine the amount of an analyte over a range of concentrations around a nominal value - the new ICH Q2 and Q14 guidelines - the Q2 gives some thought on the recommended data eg linearity = min 5 concentrations across the range, accuracy = 3 concentrations/ 3 replicates

Question 8

Range is

Answer 8

the working range of analytical method, usually representative of the accuracy, precision and linearity

Question 9

Robustness is

Answer 9

validation to include method parameters that could vary and still demonstrate control

Question 10

Method validation documentation required

Answer 10

Raise Validation Protocol – Objective / Background, Acceptance Criteria, Methodology, Validation Parameters.
This will be followed by a Validation Report - Summary of Results Vs Acceptance, Criteria, Individual Results and Discussion, Conclusions & Recommendations.

Question 11

What stats are applied during analytical method validation?

Answer 11

Two Sided T-Test can be used to demonstrate equivalence
Linearity – R value should be 0.998
Range – assay 80 – 120%
Intermediate Precision – F-Test
LOD and LOQ

Question 12

Analytical method transfer considerations

Answer 12

For multiple strength if formulation is step up and step down where placebo contrast is same any strength can be used.

If formulation is look a like which means all strength is having same average weight just active ingredient concentration is changing in such - choose lower strength where placebo is higher.

For assay and dissolution - specificity and precision to be checked
For Related substances- RS we do specificity , precision , LOQ confirmation .

If impurity is below LOQ or not detected same needs to be spiked at limit concentration and needs to prove method is capable to deter if impurity is present in sample.

Perform identification test and any TLC to avoid future challenges.

Question 13

How would you go about the method validation and tech transfer for an assay test?

Answer 13

I would raise a change control to assess the change. I would raise a tech transfer protocol and follow the guidance outlined in ICH Q2 for analytical method validation. This includes testing for the following:-
Accuracy
Precision
Specificity
LOQ /LOD
Linearity
Range

Question 14

How would you compare the data from the accuracy and precision tests? What is t value and p value, what parameters would you measure?

Answer 14

Double sided t- test
T value measures the difference between the relative variation of your sample data
The closer the T value is to 0 the less variation there is in your data
P value is the calculated probability of obtaining a t value – the larger the t value the smaller the p value

Question 15

How would you validate the method robustness?

Answer 15

Robustness typically only performed in development of a new analytical method. A number of small deliberate changes are made to method parameters such as flow rate, temperature, pH, injection volume etc. The impact on the method can

Question 16

How would you validate an analytical method transfer for an assay and what values would you expect to see?

Answer 16

Use significance tests:
T-test comparison of experimental mean with a known value (null hypothesis: no difference between experimental and known value): determine t, compare with t from tables for P = 0.05 (significance level), if tfound < tcritical null hypothesis stands, if tfound > tcritical null hypothesis is rejected

T-test comparison of means of two samples, e.g. the means from two analytical methods (null hypothesis: the two methods give the same results): determine t (using pooled std deviation), compare with t from tables for P = 0.05 (significance level), if tfound < tcritical null hypothesis stands), if tfound > tcritical null hypothesis is rejected
NOTE: comparison of means detects systematic errors

F-test for comparison of std deviations. A significance test for comparing random errors of two sets of data, e.g. whether method A is more precise than method B (one-tailed test)or whether two methods differ in their precision (two-tailed t-test). Looking at variances (i.e. squares of std deviations, F = s12 / s22, null hypothesis: variances are equal, populations normal): determine F, compare determined F with critical value from tables for P = 0.05 (significance level), for two-tailed test: if Ffound < Fcritical null hypothesis stands

Adopting the null hypothesis that the method is not subject to systematic error (→ you want the probability of random error to be “high”) i.e., that there is no difference between the observed and known values (other than that which can be attributed to random variation) → probability that the observed difference between the mean and the true value arises solely because of random errors: the lower it is the less likely that the null hypothesis is true. Usually the null hypothesis is rejected if the probability of such difference occurring by chance is less than 1 in 20 (i.e. 0.05 or 5%). The significance level is indicated P = 0.05 and is the probability of rejecting the null hypothesis.

	From Alex H:
	Data 1             Data 2
	 X                y
	Xx                yy
	Xxx            yyy
	…            …
	Mean, SD        Mean, SD,
	RSD            RSD
    Compare RSDs
    F-test
T-Test – P value greater than 0.05

Question 17

ANOVA (analysis of variance):

Answer 17

comparing more than two means
Is it just an assay method or is it a Impurities method ? – this will determine if you need LOD/LOQ

Question 18

ICHQ2 Analytical Method Validation, Annex 15 Qualification / Validation

Answer 18

Accuracy – closeness in agreement to a known standard value – assay, API and product: recovery ±2% RSD ≤ 3%; impurities: recovery ±10% RSD ≤ 10% (imp ≤ 0.5%) or ≤ 5% (imp >0.5%)
Precision - Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. Expressed as variance, standard deviation or coefficient of variation of a series of measurements. API and product: RSD ≤ 2%, Impurities: RSD < 5% (imp > 0.2%), RSD < 10% (0.1% < imp < 0.2%), intermediate precision: RSD = 1.5 x RSD for repeatability
Specificity – ability to detect content or potency of the analyte in a sample accurately
LOD/LOQ – LOD – 1 s.d from baseline, 3 s.d from baseline
LOD: 2 or 3:1 S/N or 3.3σ/S RSD ≤ 15%; LOQ: 10:1 S/N or 10σ/S RSD ≤ 20%
Linearity – obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.0.995 minimum AI r > 0.990 RSD ≤ 1.5%; impurities: r > 0.900, RSD ≤ 10% (imp ≤ 0.5%) or RSD ≤ 5% (imp > 0.5%)
Range - demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. For the assay of a drug substance or a finished (drug) product: normally from 80 to 120 percent of the test concentration;

Question 19

What range would you expect for an assay?

Answer 19

For the assay of a drug substance or a finished (drug) product: normally from 80 to 120 percent of the test concentration;

Question 20

What value would you expect for LOD?

Answer 20

3:1 signal-to-noise ratio
Below the reporting threshold for the impurity

Question 21

What value would you expect for LOQ

Answer 21

At or below the specified limit
10:1 signal-to-noise ratio

Question 22

What is the P value?

Answer 22

P Value is the probability of obtaining a particular T value

Question 23

You are carrying out a method transfer of a HPLC assay to a contract laboratory; what are your protocol expectations? What would you expect to see?

Answer 23

ONLY REPEATABILITY
Objective
Reference Standards
Test Items
Reagents
Analytical Method – Assay
Equipment
Evaluation of Analytical Data
Reporting of Results
Quality Management

Question 24

Why / when use Analytical method validation

Answer 24

• All methods should be validated
  
  • Pharmacopeia methods excluded but should be verified
  • Lab equipment must be qualified prior to method validation
  • Modification to methods should initiate revalidation
  • Validated to ICH Q2 standards

Question 25

ANALYTICAL METHOD VALIDATION (ICH Q2)

Answer 25

• Specificity - Ability to assess Specificity unequivocally the analyte
  
  • Accuracy - Closeness of agreement between the value and true value
  • Precision - Closeness of repeated measurements on same sample
  • Detection limit - The lowest amount which can be detected but not necessarily quantitated
  • Quantitation Limit - Lowest amount which can be quantitatively determined
  • Linearity - Direct proportionality between test results and concentration
  • Range - Interval between the upper and lower concentration
  • Robustness - Remain unaffected by small, but deliberate variations in method

Question 26

Why do we carry out cleaning validation? How would it be assessed and carried out?

Answer 26

To confirm effectiveness of cleaning procedure using validated methods of appropriate sensitivity
Determine toxicity
* Is the compound sensitising, mutagenic, genotoxic etc.?
* Determination of no adverse effect limit (NOAEL) for “critical effects”
* Calculation of permitted daily exposure (PDE) using NOAEL and safety factors

Perform risk assessment of manufacturing process
* Focus on product contact parts; consider non-contact
* Should establish risk reduction strategy

Consider intervals between
* Use and cleaning
* Cleaning and re-use

Bracketing of similar products and processes
* Single worst case validation study considering critical issues

Typically 3 batches

Ongoing verification

Question 27

Analytical Method Development

Answer 27

New ICH Q14

1. Understand your sample!
2. Define your method goals
3. Define analysis requirements
4. Conduct research:
   Has the analysis has been performed before.
   Previously developed methods with quantitation and sample matrices that are close to your requirements can form a starting point for your method.
5. Select Analytical Technique
6. Determine initial conditions
   e.g. Detector type, column, Mobile phase
7. Prepare sample
8. Develop the method
   Stepwise approach or Systematic screening protocols
9. Select standardisation technique
   Internal/external standards
10. Check Method performance
    SLAPRR
11. Optimise method and Check Robustness
    HPLC conditions: % organic, pH, flow rate, temperature, wavelength.
    Sample preparation: pH, shaking/sonication, sample size
    12: Validate

Question 28

Types of Methods to be Validated:

Answer 28

ID Tests
Quantitative tests for active content
Quantitative tests for impurities content
Limit tests for control of impurities

Different validation characteristics depending on method type

Test Type:
ID – Ensure ID of sample through comparison to a standard
Impurities – Quantitative/Limit test to reflect sample purity
Assay – Measure amount of sample present

Validation of Test Methods:
Validate with appropriate detection & quantification limit
Micro testing: confirm product does not impact m/org recovery
Cleanrooms  confirm sanitising agents do not impact m/org recovery

Biological Test Types:
Appearance and Description
Biological Activity
Purity and Impurities
Quantity

Question 29

Performing a Method Validation:

Answer 29

Change Control: If introducing a new method into lab
Protocol: Document validation exercise. Outlines objective and scope of exercise, all materials & equipment to be used, tests to be performed and associated acceptance criteria, action in event of failures and how exercise will be reported.
Other documentation including methods and equipment SOPs etc. should also be in place.
Report: Document conclusion of validation exercise including all results obtained.

Question 30

Analytical Method Validation parameters (8 items)

Answer 30

Specificity - the ability to assess unequivocally the analyte in the presence of components which may be expected to be present.

Linearity - the ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample
(Min of 5 Concentrations, cover range specified in Range)

Accuracy - closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.
(Min of 9 determinations over a min of 3 concentration levels covering the specified range  3 replicates per conc)

Precision – the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Three levels of precision that may be considered:

Repeatability – expresses the precision under the same operating conditions over a short interval of time. Min of 6 determinations at 100% conc.

Intermediate Precision - expresses within-laboratories variations: different days, different analysts, different equipment, etc.

Reproducibility - the precision between laboratories (collaborative studies, usually applied to standardisation of methodology). 

Range – the interval between the upper and lower concentration of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.
Assay: 80 – 120%
CU: 70 – 130%
Disso: +/-20% over specified range (taking into account spec at different time points)
IMPs: up to 120% of spec

Robustness – the measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage: pH, mobile phase composition, columns, temperature etc.

Solution Stability!
Quantitation Limit - the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. Can be visual or based on S/N 10:1

Detection Limit - the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. Can be visual or based on S/N 3 or 2:1

Question 31

Analytical Method Transfer

Answer 31

EudraLex Volume 4, Part I, Chapter 6 provides guidance
Depending of the type of method, can perform analytical method transfers by:
Joint Testing (also known as comparative testing)
A transfer where both transferring & receiving sites perform testing on same batch.
Consideration: Training before beginning method transfer.
Perform under protocol and document outcome in final report.
Results from each site compared & assessed against predetermined acceptance criteria.
For Drug Product methods where the method covers multiple different product strengths (only when DP are dose proportional), bracketing may be justified, in place of testing all product strengths (i.e. test lowest and highest strength).
Testing at each site must be performed within a suitable timeframe of each other.
Other Transfer Types Include:
Full or Partial Validation
Training
Scientific Rationale (also known as transfer waiver)
If receiving site has previously successfully completed M/T for closely related method and it can be demonstrated the methods are practically the same.
If method is a pharmacopoeial method & receiving site is familiar with technique (KF/LOD)

Method Transfer:
Transfer of analytical procedures from one lab to another
Protocol contains predefined acceptance criteria
Report documents results and outcome of method transfer
Same batch used at both labs
Useful to perform pre-transfer training exercise
Tacit knowledge NB

Analytical Method Verification:
Compendial methods must be verified to ensure lab can perform method correctly

Question 32

Impurities (ICH Q3):

Answer 32

Need to understand types and sources of impurities:
Related substances: By-products/degradation products, structurally related to drug substance
Process contaminants (chemical/physical/micro):
Process specific (reagents/solvents/catalysts), should be a consistent level
Process independent (foreign matter / equipment residues etc.)
Need to classify type of impurities and apply limits

Question 33

Comparative Dissolution Profiles

Answer 33

Showing bioequivalence: SUPAC > 50, F tests
In vitro = in glass (mimic/representation of body)
In vivo = in body
Immediate release dissolution profiles don’t give much info as drug dissolves immediately. Particle size, morphology, surface area, crystal shape, polymorphs can all impact dissolution and solubility.

Bioequivalence = Demonstrate extent and rate of absorption in two products are equiv. If bioequivalent then no need for bioequivalence studies. Typically used for generics or formulation changes.