3. Introduction
The validation of an analytical method is required to establish the
performance characteristics of the method as it is intended to
be used. Performance characteristics are expressed in terms
of analytical parameters.
Validation is an essential procedure that demonstrates that a
manufacturing process operating under defined standard
conditions is capable of consistently producing a product that
meets the established product specifications.
4. The proof of validation is obtained through the collection and
evaluation of data, preferably, beginning from the process
development phase and continuing through the production
phase.
Validation necessarily includes process qualification (the
qualification of materials, equipment, systems, buildings,
personnel), but it also includes the control on the entire process
for repeated batches or runs.
5. Objectives of validation
It reduces risk of regulatory non-compliance.
Reduction of time to the market for the new products.
Eliminates the scrap & reduces the defect cost.
Reduces the chances of product re-call from market.
It requires less in-process control & end process testing.
Parametric release of batch can be achieved in validation.
6. Parameters assesed during analytical method
validation
1. Linearity and Range
2. Specificity
3. Precision
4. Accuracy
5. Limit of Detection
6. Limit of Quantitation
7. Robustness
8. System Suitability
7. Method validation
Analytical methods used for measuring residues in cleaning
validation protocols should themselves be validated. This
validation usually means following standard industry practices for
the validation of analytical methods, including evaluation of
specificity, linearity, range, precision, accuracy, and LOD/LOQ.
8. Specificity
Specificity is a measure of the validity of the result based on
expected interferences.
In other words, one needs to confirm whether or not the method
can unequivocally measure the target species in the presence of
possible interferences.
Methods such as HPLC are generally considered specific. However,
they are only specific if possible interferences have been evaluated
to see if they change the nature of the assay.
For cleaning processes, this means that any HPLC procedure
should be evaluated to see whether possible residues from the
cleaning agent interfere with the assay.
Interferences may include changes in retention time, peak height,
or peak shape.
9. Range
Range is a series of values of the measured species or property
over which the analytical procedure was evaluated.
It is only necessary to assure that the procedure is valid over a
range of expected values.
For example, if the calculated acceptance limit for the analytical
sample is X ppm, then one might want to evaluate a range from
approximately 0.2X to 1.OX.
On the other hand, if expected results (perhaps based on
prequalification studies) are to be in the 0.1X to 0.3X range, then
validation of a range of 0.05X to 0.5X may be justified.
10. LOD / LOQ
LOD is the assay value at which it is still possible to say that the
material is present, but it may be not possible to quantify with a
specific value.
LOD is typically estimated by several techniques.
For example, for chromatographic techniques, LOD is estimated at
three times the standard deviation of a baseline response.
Values that are below the LOD are generally reported as < LOD.
11. LOQ is the lowest assay value for which a reasonable confidence
exists that the value is precise.
There are also rules of thumb for estimating LOQs. For
chromatographic procedures, the LOQ can be estimated as 10
times the standard deviation of the baseline noise.
The LOQ can also be determined experimentally; as a practical
matter, it can be considered the lower limit of the validated range
of the assay.
Any measured value below the LOQ is expressed as < LOQ.
12. Linearity
Linearity refers to the characteristic of the relationship of the
measured property to the level of analyte present.
Linearity is an indication that the measured signal is directly
proportional to the concentration of the analyte over the range.
As a general rule for cleaning validation studies, the expectations
are that assays will be linear over the range.
Estimates of linearity can be made by such techniques as
determination ~ (0.99 or better).
13. Accuracy
Accuracy refers to the trueness of the measurements to known
values. This is determined by analyzing known standards.
There is no "magic number" for acceptable accuracy. However, more
accurate methods are preferred over less accurate methods.
For example, if the acceptance criterion was 20 ppm, a method with a
accuracy of 2- 10 percent, giving a result of 18 ppm, could be
considered an acceptable result.
On the other hand, a method with an accuracy of 2- 20 percent,
giving a result of 18 ppm, will be suspect in terms of meeting the
acceptance criterion.
14. Precision
Precision refers to the reproducibility of the method and is often
measured by standard deviation.
Simple precision is the reproducibility of the results in the same lab
over a series of replicate assays using the same operator, the same
equipment, and usually on the same day.
Intermediate precision is the reproducibility of results in the same
lab using different operators, different pieces of equipment, and
generally done on different days.
Ruggedness is interlab reproducibility, involving reproducibility in
different labs
15. Keys to Method Validation
It should be noted that in many cases, preferences were given in
the discussion of specificity and accuracy.
These are not to be considered absolute. In selecting an
appropriate analytical method for the validation task, one must
balance a series of needs.
The key is to be aware of the limitations and risks associated with
any analytical method and to take steps to minimize those risks.
16. A robust cleaning procedure is one way to manage the risks
related to analytical methods and residue levels.
It should also be noted that determination of specificity, range,
linearity, LOD/LOQ, precision, and accuracy are ordinarily first
done on the analytical method itself, independent of the
sampling technique.
The sampling technique can affect the analytical method.
17. Cleaning validation
Why It Is So Important:
Pharmaceuticals can be contaminated by potentially dangerous
substances.
Particular attention should be accorded to the validation of
cleaning procedures.
Cleaning validation should be performed in order to confirm the
effectiveness of a cleaning procedure.
18. Possible contaminants:
Product residues
Cleaning agent residues and breakdown
Airborne matter
Lubricants, ancillary material
Decomposition residues
Bacteria, mould and pyrogens
19. Levels of cleaning:
Level 1 cleaning: It is used only between steps in the same
manufacturing process.
Level 2 cleaning: It is used when cleaning between steps in the same
manufacturing process. Level 2 cleaning would be used if step B was
to be performed immediately after step A for the same product line.
Level 3 cleaning: It would be performed when cleaning after an
intermediate or final product step or one product in preparation of an
intermediate step of another product.
Level 4 cleaning: It would be used after final product is ready.
20. Level 1 & Level 2 cleaning :
a) lowest risk
b) higher limits
c) less extensive cleaning
d) visual verification of clean
Level 3 & Level 4 cleaning:
a) Highest risk
b) Lower limits
c) More extensive cleaning
d) Analytical method
21. The cleaning process validation takes the
following into :
Validation of Cleaning Processes
Equipment and Personnel
Microbiological Considerations
Documentation
Sampling, Rinsing, Rinse Samples and Detergents
Establishment of Limits.
22. validation of cleaning processes
It is usually not considered acceptable to test-untilclean. This
concept involves cleaning, sampling, and testing with repetition of
this sequence until an acceptable residue limit is attained.
Raw materials sourced from different suppliers may have different
physical properties and impurity profiles. When applicable such
differences should be considered when designing cleaning
procedures, as the materials may behave differently.
23. If automated procedures are utilized (Clean-In-Place: CIP),
consideration should be given to monitoring the critical control
points and the parameters with appropriate sensors and alarm
points to ensure the process is highly controlled.
24. EQUIPMENT AND PERSONNEL
Equipment:
All processing equipment should be specifically designed to
facilitate cleanability and permit visual inspection and whenever
possible, the equipment should be made of smooth surfaces of non-
reactive materials.
Personnel:
It is difficult to validate a manual cleaning procedure (i.e. an
inherently variable/cleaning procedure). Therefore, operators
carrying out manual cleaning procedures should be adequately
trained, monitored, and periodically assessed.
25. Microbiological consideration
The existence of conditions favorable to reproduction of micro-
organisms (e.g. moisture, temperature, crevices and rough
surfaces) and the time of storage should be considered. The aim
should be to prevent excessive microbial contamination.
Equipment should be dried before storage, and under no
circumstances should stagnant water be allowed to remain in
equipment subsequent to cleaning operations.
26. The period and when appropriate, conditions of storage of
equipment before cleaning and the time between cleaning and
equipment reuse, should form part of the validation of cleaning
procedures.
This is to provide confidence that routine cleaning and storage of
equipment does not allow microbial proliferation.
27. Documentation :
Detailed cleaning procedure(s) are to be documented in SOPs.
A Cleaning Validation Protocol Should Include The Following:
The objective of the validation process
Responsibilities for performing and approving the validation study
Description of the equipment to be used.
The interval between the end of production and the beginning of
the cleaning procedure; The number of lots of the same product,
which could be manufactured during a campaign before a full
cleaning is done.
Detailed cleaning procedures to be used for each product, each
manufacturing system or each piece of equipment.
28. The number of cleaning cycles to be performed consecutively.
Sampling procedures, including the rationale for why a certain
sampling method is used.
Clearly defined sampling locations.
A Final Validation Report should be prepared. The conclusions of
this report should state that cleaning process has been validated
successfully.
The report should be approved by the Plant Management.
29. SAMPLING, RINSING, RINSE SAMPLES AND DETERGENTS
Sampling:
There are two general types of sampling that are considered
to be acceptable, direct surface sampling (swab method) and indirect
sampling (use of rinse solutions). A combination of the two methods
is generally the most desirable.
Detergents:
Detergents should be easily removable, being used to
facilitate the cleaning during the cleaning process.
When detergents are used in the cleaning process, their
composition should be known to the user and their removal should
be demonstrated.
Acceptable limits should be defined for detergent residues
after cleaning.
30. Last Rinse:
Water for injection should be used as the last rinse for
product-contact equipment to be utilized in the fabrication of
sterile products.
Purified water is considered acceptable as the last rinse for
product-contact equipment used in the fabrication of non-sterile
products or sterile products for ophthalmic use.
31. Establishment of limits
The pharmaceutical company's rationale for selecting limits for
product residues should be logically based on a consideration of
the materials involved and their therapeutic dose.
The limits should be practical, achievable and verifiable.
32. Typical analytical procedures
Below is a short listing of appropriate analytical procedures and their
applicability for cleaning validation purposes.
This list is not meant to be exhaustive. High Performance Liquid
Chromatography (HPLC) involves injection of the sample into a
chromatographic column, separation of the target species from other
components in the sample, and then measurement of that target
species as it exits the column by ultraviolet (UV) spectroscopy,
conductivity, or ELSD (evaporative light-scattering detection).
HPLC can generally be tweaked such that it is specific for the target
species. The equipment is generally available in pharmaceutical
facilities.
33. Total Organic Carbon
TOC involves oxidation of the sample (by any of a variety of
techniques) and measurement of the carbon dioxide generated by
either infrared spectrometry or conductance.
The method is generally considered nonspecific.
TOC usually involves an assumption that all of the measured
carbon is due to the target species, and the maximum possible level
of the target species is calculated based on this assumption.
34. TOC is becoming more widely used because it is an acceptable
technique to replace for the oxidizable substances test for USP
Purified Water and because of the possible degradation of actives
due to the cleaning environment.
For the latter reason, TOC is used commonly in the biotechnology
industry for cleaning validation purposes.
35. Atomic Absorption
Atomic absorption is a specific method for metal ions. It can be
utilized in the determination, for example, of sodium and/or
potassium that may be present in cleaning formulations.
This is not necessarily a common instrument in pharmaceutical
analytical laboratories.
36. Ion Chromatography
Ion chromatography includes specific methods for both anions and
cations in cleaning formulations.
It can be used to measure both sodium and potassium as cations,
and different methods can be used to separate and measure
anions, such a s the anions from acidic detergents (phosphates,
citrates, glycolates) or builders (carbonates, gluconates, silicates,
EDTA [ethylenediaminetetraacetic acid]).
This is not necessarily a common instrument in pharmaceutical
analytical laboratories, but it is becoming more widely used.
37. Ultraviolet Spectroscopy
For certain surfactants that have a chromophore, UV spectroscopy
can be an acceptable tool.
The instrumentation is readily available in many pharmaceutical
analytical laboratories.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is commonly used in the analysis of protein for the
determination of actives.
However, because proteins are usually degraded by the harsh
conditions (temperature and pH) of the cleaning environment,
ELISA has limited practical use for cleaning validation studies.
38. Titrations
Titrations can vary from alkalinity or acidity titrations, which can
be used to give upper level estimates of cleaning agents present, to
more specific titration procedures to measure components of
cleaning agents, such as titrations for chelants in cleaning agents.
The laboratory equipment for these procedures is generally readily
available.
39. Conductance
Conductivity measures a nonspecific property of ions in solution.
It can be used as an upper limit estimate of the amount of an
alkaline or an acid cleaning agent.
Dilute solutions exhibit a linear behavior. If not available, the
equipment can be purchased relatively inexpensively.
40. Some companies have tried to use pH a s an estimate of residues of
either an alkaline or an acidic cleaning agent.
pH can be a useful monitoring tool in that a high or low pH can
indicate a system out of control.
However, it is not a preferred technique for determining actual
levels of alkaline or acidic residues.
41. References
[1] FDA. 1998. Human drug cGMP notes, vol. 6, no. 4. Rockville, Md., USA:
Food and Drug Administration, Center for Drug Evaluation and Research.
[2] Gavlick, W. K., L. A. Ohlemeier, H. J. Kaiser. Pharmaceutical Technology.
[3] FDA. 1993. Guide to inspections of validation of cleaning processes.
Rockville, Md., USA: Food and Drug Administration, Office of Regulatory
Affairs.
[4] LeBlanc, D. A. 1998. Pharmaceutical Technology.
[5] ICH. 1995. Guideline for industry: Text on validation of analytical
procedures.
[6] ICH. 1997. Guideline on the validation of analytical procedures:
Methodology..
