ANALYTICAL METHOD,CLEANING VALIDATION

BY : SAI VIVEK KOSARAJU, I M.PHARM, PHARMACEUTICS

JSS COLLEGE OF PHARMACY,OOTY.

ANALYTICAL METHOD VALIDATION

VALIDATION

Validation is defined as the “FINDING OR TESTING THE TRUTH OF SOME THING.”

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

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

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.

PARAMETERS ASSESED DURING ANALYTICAL METHOD VALIDATION

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1. Linearity and Range

2. Specificity

3. Precision

4. Accuracy

5. Limit of Detection

6. Limit of Quantitation

7. Robustness

8. System Suitability

Linearity and Range

The range of an analytical procedure is the interval between the upper and lower levels of analyte (including these levels) that have been demonstrated to be determined with a suitable level of precision, accuracy, and linearity,

For establishment of linearity, minimum 5 concentrations are recommended.

Linearity results should be established by appropriate statistical methods.

LINEARITY FOR CONC vs RESPONSE

Conc.(µg/ml)

√Response

1 0.25

2 0.5

3 0.75

4 0.96

5 1.25

If linearity is not attainable, a nonlinear model may be used. The goal is to have a model (whether linear or nonlinear) that describes closely the concentration-response relationship

The following parameters should be determined:

correlation coefficient y-intercept slope of the regression line

The range of the procedure is validated by verifying that the analytical procedure provides acceptable precision, accuracy, and linearity when applied to samples containing analyte at the extremes of the range as well as within the range.

Precision

The precision of an analytical procedure expresses 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

concentration

Absorbance

10µg/ml

mean

0.210.320.390.530.36

Less Variation More Variation

High Precision Low Precision

Precision may be considered at three levels:

PrecisionRepeatabili

ty

Intermediate

Precision

Reproducibility

1. Repeatability Repeatability expresses the precision under the same

operating conditions over a short interval of time. Repeatability should be assessed using a minimum of

9 determinations covering the specified range.

2. Intermediate Precision Intermediate precision expresses variations within

laboratories, such as different days, different analysts, different equipment, and so forth

Reproducibility Reproducibility expresses the precision

between laboratories. It is assessed by means of an inter-laboratory trial. (Defined as ruggedness in USP, ISO 17025)

Accuracy

Closeness of agreement between the conventional true value / an accepted reference value and the value found

High Accuracy Less Accuracy

(Less Precision) (High Precision)

ASSESMENT OF ACCURACY

Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range (e.g., 3 concentrations/3 replicates each of the total analytical procedure).

Accuracy should be reported as percent recovery by the assay of known added amount of analyte in the sample or as the difference between the mean and the accepted true value.

LIMIT OF DETECTION & LIMIT OF QUANTITATION

Limit of Detection:• It is the lowest amount of analyte in a sample

which can be detected but not necessarily quantitated.

Limit of Quantitation:• It is the lowest amount of analyte in a sample

which can be quantitatively determined with suitable precision and accuracy.

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Determination of LOD & LOQ

Method Based on visual

evaluation

Based on standard deviation of response and slope

LOD = 3.3 σ / Slope

Signal to noise ratio 2:1 or 3:1

Method Based on visual

evaluation

Based on standard deviation of response and slope

LOD = 10 σ / Slope

Signal to noise ratio 10:1

• Limit of Detection • Limit of Quantitation

SPECIFICITY

The ability to detect the analyte in the presence of interfering substances (typically impurities, degradants, matrix)is called as specificity.

1)Identification Suitable identification tests should be able

to discriminate between compounds of closely related structures which are likely to be present.

The discrimination of a procedure may be confirmed by obtaining positive results from samples containing the analyte, coupled with negative results from samples which do not contain the analyte.

The identification test may be applied to materials structurally similar to or closely related to the analyte to confirm that a positive response is not obtained.

2. Assay and impurity test:a. Impurities are available For the assay , this should involve

demonstration of the discrimination of the analyte in the presence of impurities and/or excipients.

This can be done by spiking pure substances with appropriate levels of impurities and/or excipients and demonstrating that the assay result is unaffected by the presence of these materials

For the impurity test, the discrimination may be established by spiking drug substance or drug product with appropriate levels of impurities and demonstrating the separation of these impurities individually and/or from other components in the sample matrix.

b. Impurities are not available If impurity or degradation product standards are

unavailable, specificity may be demonstrated by comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure e.g. pharmacopoeial method or other validated analytical procedure.

As appropriate, this should include samples stored under relevant stress conditions: light, heat, humidity, acid/base hydrolysis and oxidation

Robustness

The robustness of an analytical procedure is a 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.

If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure, such as:

• Use solution within 24 hours• Maintain temperature below 25 degrees

In the case of liquid chromatography, examples of typical variations are:

influence of variations of pH in a mobile phase influence of variations in mobile phase composition different columns (different lots and/or suppliers) temperature flow rate In the case of gas-chromatography, examples of typical

variations are: different columns (different lots and/or suppliers) temperature flow rate

System Suitability

System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such.

CLEANING VALIDATION

WHY CLEANING VALIDATION 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.

Possible contaminants: Product residues Cleaning agent residues and breakdown Airborne matter Lubricants, ancillary material Decomposition residues Bacteria, mould and pyrogens

LEVELS OFCLEANING :

There are 4 levels of cleaning, they are: a) Level 1 b) Level 2 c) Level 3 d) Level 4Level 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.

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

THE CLEANING PROCESS VALIDATION TAKES THE FOLLOWING INTO ACCOUNT:

Validation of Cleaning Processes, Equipment and Personnel, Microbiological Considerations, Documentation, Sampling, Rinsing, Rinse Samples and

Detergents, Establishment of Limits.

VALIDATION OF CLEANING PROCESSES

It is usually not considered acceptable to test-until-clean. 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.

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.

During a campaign (production of several batches of the same product), cleaning between batches may be reduced. The number of lots of the same product which could be manufactured before a complete/ full cleaning is done should be determined

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.

MICROBIOLOGICAL CONSIDERATIONS

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.

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.

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;

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.

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

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.

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