Validation

Cleaning Method Validation Protocol for Pharmaceutical Equipments Protocol for the validation of the cleaning validation of the pharmaceutical manufacturing equipments. Objective:

The purpose of the study is to validate analytical method for determination

of traces of API contents in Swab & Rinse samples and to establish

documented evidence and provide the procedure for the same.

Scope:

The scope should evaluate the acceptability of

an analytical method for determination of traces of API contents in swab and

rinse samples by HPLC. This protocol should define the procedure,

documentation, references, acceptance criteria and results evaluated for

determination of traces of API contents in Swab & Rinse samples by HPLC.

Responsibilities:

Analytical R&D Department:

Quality Control Department:

Regulatory Affairs:

Quality Assurance Department:

Validation Team & Approval:

It should include the approval of the individuals who have performed the

validation study, supervised the validation, completed the records,

performed the testing of the product and maintained the equipment.

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Training:

Training should be given to the concerned persons and training record

should be maintained.

Details of Analytical Method Procedure and Solution Preparations:

Test Procedure:

The detailed test procedure is as per IP/BP/USP/IH

Description: Generic Name of the Product

Determination: By HPLC

Reagents Required :

Raw material / Working Standard/Impurities:

Name of API

Placebo

Apparatus:

Clean and Dry Class ‘A’ glassware: Volumetric flasks, Bulb Pipette, swab, test

tube SS plate (10 X 10 cm SS 316L plate)

Preparation of Buffer Solution :

Mobile Phase Preparation:

Chromatographic Conditions:

Instrument :

Column :

Wavelength :

Flow rate :

Column Temperature :

Injection Volume :

Run Time :

Diluent :

Preparation of Solutions:

Blank Solution Preparation (Diluent):

Standard Solution Preparation :

Preparation of Cleaning Sample Solutions:

For Swab Samples:

Soak the swab sample in 5.0 mL of diluent. Sonicate for about 10 min. Filter

the solution through 0.45 μ syringe filter and inject into the HPLC system.

For Rinse Samples:

Filter the rinse samples through 0.45 μ syringe filter, discard first few mL, fill

the HPLC vials and inject into the HPLC system.

Procedure:

Equilibrate the column with mobile phase for 30-45 minutes. Separately

inject blank (single injection), standard solution (five Injections) and sample

preparations (single injection) and record the response of the principal peak

& other related peaks.

System Suitability:

1. Inject the blank solution. Disregard any peak due to blank solution in the test solution.2. The RSD of peak area of API in five replicate injections of standard solution should not be more that 2.0%.Calculations:

Calculate the amount of API present in ppm in the rinse sample using

the following formula.

Where,

AT = Area of API in Rinse Sample Solution

As = Average Area of API in Standard Solution

Ws = Weight of standard taken (mg)

Ds = Dilution of standard

P = Purity of API on as is basis

Calculate the amount of API present in the swab sample using the following

Formula.

Where,

AT = Area of API in Swab Sample Solution



Ds = Dilution of standard

V = Final volume of swab sample (5 mL)

P = Purity of API on as is basis

Acceptance Criteria:

The amount of API recovered from the effective surface area of equipment

should not be more than 10 ppm to take the subsequent batches.

Analytical Method Validation Parameters:

Analytical Method for determination of Traces of API in swab & rinse samples

should be validated for following parameters:

System Suitability & System Precision:

Objective

To demonstrate and verify that the system suitability parameters of the

chromatographic system are adequate for the subjected analysis.

Procedure

Prepare Standard solution of API at its target concentration (10 ppm) &

other solutions as per test procedure. Inject blank, five replicates of standard

solution. Calculate RSD for area of API peak from five replicates of standard

solution. Record the results in observation Table-1

Observations: Table- 1 (HPLC ID )

Standard Solution (10 ppm)

Sr. No. Area of API

1

2

3

4

5

Average

SD

RSD (%)


The RSD of peak area of API in five replicate injections of standard solution

should not be more than 2.0%.

Linearity & Range:

Objective

To demonstrate that the analytical method is capable to:

• Obtain test results, which are directly proportional to the concentration

(amount) of analyte in the sample. (Linearity)

• Provide acceptable degree of linearity and precision when applied to

samples containing amounts of analyte within or at the extremes of the

specified range of the analytical procedure (Range).

LOD & LOQ Determination:

Procedure:

Prepare a series of standard preparations (minimum five concentrations) of

API over a range starting from (i.e.0.1% of standard concentration). Inject

each of the standard preparation in triplicate and then take average area for

calculations. Plot a graph of concentration vs peak area to establish LOD &

LOQ of API by regression function.

Plot a linearity graph of a concentration (ppm) verses average area at each

level and determine the slope. The slope S may be estimated from the

calibration curve of the analyte. The estimate of σ may be carried out based

on the calibration curve. Record the observation in Table-2A & Table-2B.

Determine the LOD and LOQ concentration level from the following formula.

The Limit of Detection (LOD) may be expressed as:

LOD = 3.3 σ

S

The Limit of Quantitation (LOQ) may be expressed as:

LOQ = 10 σ

S

Where σ = The residual SD of the regression line.

S= The slope of the calibration curve.

Observations: Table- 2 A (HPLC ID )

Standard Solution (10 ppm)

Sr. No. Area of API

1

2

3

4

5

Average

SD

RSD (%)

Observations: Table 2B (HPLC ID )

Standard Solution

Sr

.

N

o.

Concentration (ppm) Average Area of API

1

2

3

4

5

6

7

8

9

10

Correlation Coefficient ‘r’

Slope (m)

Y intercept


1. The system suitability criteria should pass as per analytical method.2. Derive the concentration for LOD & LOQ level from linearity studies.3. The Correlation Coefficient ‘r’ should not be less than 0.995.Precision at LOQ Level:

Inject LOQ level in six replicates to perform the precision at LOQ level.

Record the results in observation Table-2C.

Observations: Table 2C (HPLC ID )


The RSD of six injections at LOQ level

should not be more than 10.0 %.

Linearity from LOQ to 200%:

Procedure

To evaluate linearity, prepare

standard solutions of API of six

different concentrations (i.e. LOQ to

200% of target concentration). Inject each of the standard concentration in

triplicate. Plot a linearity graph of concentration (ppm) verses average area

at each level. Calculate the correlation coefficient, slope (m), Y intercept and

record the observations in Table-2D & Table-2E as given below.

Observations: Table 2D (HPLC ID )

Sr.

No.

LOQ

Level Area of API

1Injection_

1

2Injection_

2

3Injection_

3

4Injection_

4

5Injection_

5

6Injection_

6

Average

SD

RSD (%)

Standard Solution (10

ppm)

Sr. No. Area of

API

1

2

3

4

5

Average

SD

RSD (%)

Observations: Table 2E (HPLC ID )

From Standard Solution

Sr.N

o.

Linearity Level

(%)Concentration (ppm) Average Area

1 LOQ

2 25

3 50

4 75

5 100

6 125

7 150

8 200

Correlation Coefficient ‘r’

Slope (m)

Y intercept


1. The system suitability criteria should pass as per analytical method.2. Correlation Coefficient ‘r’ should not be less than 0.995.Accuracy/Recovery:

Objective

To demonstrate that the analytical method is capable to yield data values

close to true values. This described as follows.

• Accepted as a conventional true value. (Accuracy)

• An accepted reference value and the value found. (Recovery)

Recovery on Stainless Steel Plate (10 X 10 cm SS 316L plate):

Preparation of Stock Solution:

Preparation of Standard:

Establishment of Recovery Factor:

Prepare standard stock solution of the drug substance in the selected solvent

and spike separately 0.5 mL for 50%, 1 mL for 100% and 1.5 mL for 150%

respectively on the 10 cm x 10 cm SS 316 L plate and disperse evenly.

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Allow the plate to dry and perform swabbing. Transfer 5 mL of diluent to a

cleaned test tube. Place a clean swab into the test tube. The swabbing

should be done so as to cover the entire surface by making perpendicular

vertical and horizontal strikes. Care should be taken to lift the swab at the

end of each strike made on the SS plate. Collect the total swabbings into the

test tube containing 5 mL of diluent and sonicate for at least 10 minutes and

dilute to 10 mL with diluent. The concentration of test solution should be

close as that of target standard concentration. Filter the solution through

0.45µ syringe filter. Similarly, prepare controlled blank by dipping the swab

stick in 5 mL diluent & sonicating it for 10 min. Then, inject diluent blank,

controlled blank, standard and test solution into the chromatographic

system.

Calculate the Amount of residue recovered “in ppm”.

Where,

AT = Area of API in Swab Sample Solution



D1 = Dilution Factor of standard

V f = Final volume of sample

Vs = Volume of Standard spread on the 10x10 cm SS plate.

Calculate the % recovery

Rp = Amount of residue recovered “in ppm”

RT = Amount of residue added “in ppm”

[RF = Recovery Factor (RF= 100/ % Recovery of API from SS plate)]

Procedure:

Inject blank, controlled blank &standard solution and each recovery level as

per following sequence. Record the results in observation Table-3A & Table-

3B.

Details

No. of injections

Blank 1Controlled Blank 1Standard solution 5Recovery level -1 (50%) 3Recovery level -2 (100%) 3Recovery level -3 (150%) 3Standard solution (Bracketing) 1

Observations: Table 3A (HPLC ID )


ppm)

Sr. No. Area of API

1

2

3

4

5

Average

SD

RSD (%)


Standard Solution

Sr.N

o.

Replicates Proposed concentration

taken on plate (% of target

concentration)

Recovery

(%)

1

1 502 503 50

21 1002 1003 100

31 150

2 1503 150

Average percentage recovery: Acceptance criteria:1. The system suitability criteria should pass as per analytical method2. The recovery from SS plate in residue analysis should be between 70 % -130 %. Placebo Recovery :

Objective

To demonstrate that the analytical method is capable to yield data values

close to true values. This described as follows.

• Accepted as a conventional true value. (Accuracy)

• An accepted reference value and the value found. (Recovery)

Procedure

Determine the accuracy/ recovery by spiking the drug substance in placebo

at LOQ, 50 %,

100 % & 150 % levels of target concentration of API (i.e. 50 % - 150% of 10

ppm) respectively. Add drug amount at LOQ, 50%, 100%, and 150% levels to

the placebo respectively. Prepare each concentration in triplicate & inject

once. Calculate and record the percentage recovery of API in observation

Table-4A, Table-4B &Table-4C.



ppm)

Sr. No. Area of API

1

2

3

4

5

Average

SD

RSD (%)


Level

Amount ofPlacebo

Amount of std. addedX

Response

Amount RecoveredY

X2 XY

LOQ level

-- --

Formulae: n (SXY) - (SX) (SY ) % R =------------------------------------ X 100 n (SX2 ) - (SX )2

Where ‘n’= No. of experiments i.e. 3

Observations: Table 4C (HPLC ID )

Level

Amount of

Placebo

Amount of std. added

X

ResponseAmount

RecoveredY

X2 XY

50% -- --

Mean

100%

-- --

Mean

150%

-- --

MeanSX = SY = SX2 = SXY

=

Formulae: n (SXY) - (SX ) ( SY ) % R =------------------------------------ X 100 n (SX2 ) - (SX )2

Where ‘n’= No. of experiments i.e. 9Acceptance Criteria:

1. The system suitability criteria should pass as per analytical method. 2. The average percent recovery at LOQ level should be between 80%-120%3. Average percent recovery from 50% to 150% should be within 90% to 110% of target concentration (theoretical value).Solution Stability:

Objective

To determine the stability of swab sample solution.

For Swab Sample Solution

Prepare the swab sample solution at 100 % level as per the test procedure in

‘Recovery on SS plate’ parameter (refer section 8.0), and inject the solution

at zero hour. Place the same solution at room temperature (22ºC ± 2ºC) and

inject after 2, 4, 8, 12 and 24 hrs. Calculate % recovery at different time

intervals upto 24 hrs. Record the observations in observation Table-5A &

Table-5B.



ppm)

Sr. No. Area of

API

1

2

3

4

5

Average

SD

RSD (%)

Observations: Table-5B (HPLC ID )

Swab Sample

Solution

Time in hrs%

Recovery

Initial

2

4

8

12

24

Average

SD

RSD (%)


1. The system suitability criteria should pass as per analytical method.

2. The RSD of % recovery on SS plate at different time intervals up to 24 hrs

should not be more than 5.0

Filter Evaluation:

Objective

To demonstrate that the analytical method is unaffected by using filtration

technique as well as by centrifuging the sample solutions.

Procedure

Prepare the sample at 100 % level as per test procedure in ‘Placebo

Recovery’ parameter (refer section 8.0). Filter the sample through 0.45 µm

syringe filter. Centrifuge the same sample at 2000 rpm for 15 minutes & also

filter the same sample through Whatman filter paper No. 1. Record the

results in observation Table-6A & Table-6B.

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Observations: Table-6A (HPLC ID )


ppm)

Sr. No. Area of API

1

2

3

4

5

Average

SD

RSD (%)

Observations: Table-6B (HPLC ID )

Type of filter /centrifuge % RecoveryAbsolute

Difference

0.45µ syringe filter Centrifuge Whatman No. 1 filter paper


1. The system suitability criteria should pass as per analytical method.2. The absolute difference between % recovery of sample filtered through 0.45 µ syringe filter and unfiltered (centrifuged) sample should not be more

than 2.0.

3. The absolute difference between % recovery of sample filtered through Whatman No.1 filter and unfiltered (centrifuged) sample should not be more

than 2.0.

4. The filtered samples should not show any extraneous peak as compared to unfiltered (centrifuged) samples.Recommendations and Suggestions:

Record the recommendations or suggestions based on the interpretation of

the results and reference documents where required in the validation report.

Revalidation Criteria:

Revalidation may be necessary in the following circumstances:

- Changes in the synthesis of the drug substance;

- Changes in the composition of the finished product;

- Changes in the analytical procedure.

The degree of revalidation required depends on the nature of the changes.

Annexure:

Annexure should be attached to the validation report.

Reference:

Abbreviations:

No. Number+ Plus or minusi.e. That is

RSD

RRF

%

Relative Standard Deviation

Relative retention factor

PercentageID

SS

WS

ppm

hrs

API

Identification number

Stainless Steel

Working Standard

Parts per million

Hours

Active pharmaceutical ingredient

Revision History:

Sr. No. Revision No. Effective Date Change Control No.

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