EXPLICACIÓN DE VALIDACIÓN DE LIMPIEZA

7/30/2019 EXPLICACIN DE VALIDACIN DE LIMPIEZA

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Development and Validation of

Analytical Test Methods for

Cleaning Samples

Presented by: Rafail Usatinsky,

Cedarburg Hauser Pharmaceuticals

July 2010

Cleaning Validation

Cleaning validation is a validation program to

verify that the procedures used to clean product

residue from process equipment and components,

will consistently and significantly reduce the

amount of active and/or excipients and cleaning

agents to a concentration within calculated

acceptance limits


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Potential Contaminants

Previous API

Precursors to the API

By-products and/or degradation products of the API

Solvents and other materials employed during the

manufacturing process

Micro-organisms

Cleaning agents and lubricants*Reference: APIC, Guide to Cleaning Validation in API plants, 1999

Elements of Cleaning Validation

Establishment of acceptance criteria

Cleaning procedure

Identification of the equipment

Characterization of the products (activity/toxicity,

solubility)

determination and characterization of the cleaning agents

Sampling Procedure and its validation

Analytical method and its validation

Validation protocol

Validation report


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Acceptance Criteria

Option A: Limiting the level based on toxicity data (Acceptable

Daily Intake (ADI) is calculated with suitable safety factors

applied and this is converted to the maximum allowable

carryover to the API.

Option B: Pharmacological Dose Method (reduce the levels of

residual product to no greater than 1/1000 of normal

therapeutic dose of the next product).

Option C: Limiting the level of product which could appear in

the following products from 10 ppm up to 0.1%.

Sampling Procedures

Swab sampling

Does not cover the entire equipment surface

(check worst case location)

Determine swabbing efficiency (% recovery)

Ensure that extractables of the swab do not

interfere with the sampling method


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Sampling Procedures

Rinse sampling

Covers the entire surface area

Ensure chosen solvent has appropriate recovery

Easier to sample

Reduced number of samples

Needle in the Hay?


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Analytical Methods

Ability to detect the target substance at

acceptance criteria levels (LOQ/LOD)

Ability to detect the target substance in

presence of other materials (Selectivity)

Use of non-specific methods for contaminants

determination (such as TOC)

Multi-disciplinary Team

Production unit is responsible for review and

checking equipment associated with the product.

R&D unit is responsible for review and checking

cleaning procedures and rinse solvents associated

with the product.

QC or Analytical Services group is responsible foranalytical method development and validation.

QA is responsible for review and authorization of

documentation associated with cleaning validation.


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Solvent Selection

DI water

Remove cleaning agent

Remove water soluble salts

Conductivity test, USP ( 10 S)

pH test USP (4 pH 8)

Organic solvent

Dissolve primary contaminants

Easy to dry equipment/evaporate for analysis

Use for multiple products

Direct sample analysis

Select Worst-Case Product for Cleaning

(Solubility Study in Methanol)

Purpose

Evaluate effectiveness of using methanol as a cleaning solvent in reactors,

drying trays, lines, etc. used in the production of GMP materials.

Employ generic HPLC methods along with a specific UV max for each compound.

Study Design

Place 200 mg sample in a scintillation vial along with 5mL of methanol and stir

overnight at room temperature.

If a clear solution resulted the solubility > 40 mg/mL defined as completely

soluble.

If the material is not completely soluble, take an aliquot of the supernatant,

filter through a 0.45 m syringe filter, dilute and compare to a standard

solution of known concentration (0.1 - 1.0 mg/mL).


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Solubility in Methanol (contd)

Many of the supplied samples were highly soluble in methanol (> 40 mg /

mL), and for these compounds methanol represents an excellent choice as

a cleaning solvent.

For those compounds where the solubility was < 10 mg / mL, either other

solvents should be investigated for cleaning, or multiple rinses with

methanol should be performed to ensure thorough cleaning of the

equipment. A comparison between the first and second rinse, and the

second and third rinse, etc. would show whether the substance was still

being eluted from the contact surfaces.

Compounds with a solubility > 10 mg / mL but < 40 mg / mL represent agray area and should be the subject of further discussion as to developing

a more effective cleaning method.

Chart of Solubility of Compounds Classified as Minimally-Moderately Soluble in Methanol(< 10 mg/mL = Minimally Soluble; 10-40 mg/mL = Moderately Soluble)

Compounds Found to be Highly Soluble (> 40 mg/m L) were not I ncluded in Chart

0

5

10

15

20

25

30

35

40

GMP Compounds

SolubilityinMethanol(mg/mL)

Compound 1Compound 2Compound 3Compound 4Compound 5Compound 6Compound 7)Compound 8Compound 9Compound 10Compound 11Compound 12Compound 13


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Maximum Allowable Carryover (MAC)and Acceptance Limits

RINSE

Residue limit is 0.002% =>

20 mg for 1kg batch

Typical final rinse is 10L =>

0.002 mg/mL residue

concentration

Limit for rinse = 0.002mg/mL

SWAB

Residue Limit = 20 mg

Limit for swab =

(MAC/Total Surface Area x Swab

Area)/ Volume of Swab solution

Example: (20mg/40000cm2 x

100 cm2)/ 10 mL = 0.005 mg/mL

Method Development

Not different from general approach

Determine detection technique

Strong UV absorption consider HPLC-UV

Low UV absorption HPLC-CAD, TLC

Concentrate sample to increase sensitivity

Select swabs


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Method and Detection SelectionCompound Structure Method Validation

Fentanyl HPLC-UV

Analytical Validation

Rinse/Swab Recovery

Oxandrolone TLC


Rinse/Swab Recovery

Inorganic Product

Sodium Containing

Compound Conductivity


Rinse/Swab Recovery

N N

CH3

O

O

O

OH

H

HPLC Method Development

Compound: Fentanyl

Column: Phenomenex Luna C18, 4.6 x 150mm, 5 um

Mobile phase: 0.14% HClO4: ACN = 65:35

Flow rate: 1.2 mL/min

Detection: UV at 206 nm

Injection volume: 5 uL Column Temperature: 30C

Run time: 10 min (Fentanyl RT = 6 min)

Sample preparation: dilute methanol rinse or swab sample

with water 1:1 ratio


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HPLC Method Development Defininga Game Plan

Retention > Selectivity > Sensitivity

Select starting conditions (use assay method)

Getting retention right (short run time)

Variables that affect selectivity (rinse solvent,

swabs leachables)

Sample diluent (avoid split peaks) Adjust sensitivity as necessary (target LOQ)

Standard HPLC

Std UV

nm200 210 220 230 240 250 260 270 280 290

Std at 206, 220, 254 nm

min1 2 3 4 5 6 7 8 9

mAU

-5

0

5

10

15

20

25

30

DAD1A, Sig=206,4Ref=360,100(CLEANING\10062107.D)

5

.3

5

8

DAD1B, Sig=220,4Ref=360,100(CLEANING\10062107.D)

5

.3

5

8

DAD1C, Sig=254,4Ref=360,100(CLEANING\10062107.D)

5

.3

5

8


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Solvent Grade

Methanol ACS Grade

min0 1 2 3 4 5 6 7 8 9

mAU

-10

0

10

20

30

DAD1A, Sig=206,4Ref=360,100(CLEANING\10062108.D)DAD1B, Sig=220,4Ref=360,100(CLEANING\10062108.D)DAD1C,Si g=254,4Ref=360,100(CLEANING\10062108.D)

Methanol Tech Grade

min0 1 2 3 4 5 6 7 8 9

mAU

-10

0

10

20

30

DAD1A,Sig=206,4Ref=360,100(CLEANING\10062109.D)

5

.2

0

9

DAD1B,Sig=220,4Ref=360,100(CLEANING\10062109.D)DAD1C,Sig=254,4Ref=360,100(CLEANING\10062109.D)

Diluent (Split Peak)

100% Methanol 50% Methanol

m0 1 2 3 4 5 6

mAU

0

50

100

150

200

250

300

350

400

VWD1 A, Wavelength=246 nm (30202\10051201.D)

3

.428-Analyte

m0 1 2 3 4 5 6

mAU

0

20

40

60

80


3.4

25-Analyte


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Swabs Extractables

Swab Recovery Blank Swab Extracted (2 inj)

0 1 2 3 4 5 6 7 8 9

mAU

-10

-5

0

5

10

15

20

25

30

35


5.1

89-

Analyte

m0 1 2 3 4 5 6 7 8 9

mAU

-10

-5

0

5

10

15

20

25

30

35


m0 1 2 3 4 5 6 7 8 9

mAU

-10

-5

0

5

10

15

20

25

30

35


6.4

95-

Swabextractable

Cleaning Validation

Validation of analytical method: typical

method validation parameters (linearity,

accuracy, precision, specificity, robustness)

Validation of rinse recovery from different

surfaces (coated glass, stainless steel, teflon) Validation of swabs recovery


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Method Validation (Linearity)

The linearity of the test method

was determined by analyzing

triplicate injections of Fentanyl at

five concentrations of

approximately 1, 3, 5, 7 and 10

g/mL. The five-level calibration

curve was found to be linear with

correlation coefficient (r) of

0.995.

Fentanyl Linearity

R2 = 0.9999

0.00000

50.00000

100.00000

150.00000

200.00000

250.00000

300.00000

350.00000

0.0000 2.0000 4.0000 6.0000 8.0000 10.0000 12.0000

ug/ml

area

Method Validation

(Accuracy/Precision)

Sample ID InjectionTheory

mg/mLPeak Area

Exp.

mg/mL% Recovery

% Recovery

Averages

1 g/mL

1 0.00109 33.47281 0.001 102.629

101.8492 0.00109 33.18301 0.001 101.334

3 0.00109 33.26467 0.001 101.584

5 g/mL

1 0.00543 163.56015 0.005 99.896

99.7952 0.00543 163.24034 0.005 99.701

3 0.00543 163.38344 0.005 99.788

10 g/mL

1 0.01085 330.45963 0.011 100.916

100.8232 0.01085 329.64816 0.011 100.668

3 0.01085 330.35934 0.011 100.885

Average (1, 5, 10 g/mL) 100.822

Std. Deviation9 0.957

% RSD9 0.949


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Method Validation (System Suitability,Specificity/Sensitivity)

System suitability consisted of 6 consecutive injections of

working standard (%RSD injection precision).

Specificity of the method was determined by comparing

diluent blank injections with sample injections.

The signal-to-noise ratio (S/N) of analyte at the targeted 1

g/mL LOQ concentration was determined.

The detection limit was calculated using the determined S/N

at 1 g/mL as follows:

LOD, g/mL = Conc. of Analyte, g/mL x 3

S/N

Method Validation SummaryValidation Parameters Test Parameters Limits Results

Sys tem Suit abilit y % RSD6 working standard 5% 0.2%

Method Linearity

(1 to 10 g/mL)

Correlation coefficient (r) 0.995 1.000

y-Intercept Informational -0.01227

Slope Informational 30.36423

Residual standard

deviationIn fo rmat iona l 0.86422

Method Accuracy (1, 5, 10

g/mL)% Recovery 80 120% 101%

Method Precision

(1, 5, 10 g/mL) % RSD9 10% 1%

Specificity Blank injectionsNo interfering

peaks

No interfering

peaks

Method Quant itat ion Limit S/N at 1 g/mL 10 22

Method Detec tion Limit Concent ra tion at S/N = 3 Repor t g/mL 0.15


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Rinse Recovery Study (Flow Diagram)

Clean Coupons

(cleaning solution

and DI water)

Prepare Stock

Solution

(in methanol)

Spike coupon (above and

below acceptance limit)

Rinse Coupon

(with methanol)

Analyze samples

(dilute or concentrate

as needed)

Rinsing Coupon


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Rinse Recovery Study

Conduct for the specific product to be tested on the production

equipment.

Determine the recovery and repeatability of the rinsing analysis from the

equipment surfaces.

If recovery results do not meet the acceptance criteria a different

solvent, or a larger rinsing volume may need to be used

Rinse recovery studies are performed by spiking stainless steel, and/or

coated glass, and or teflon plates.

Product is evenly distributed onto the plate at concentrations above and

below the acceptance criteria.

Repeat the procedure without active residue to address the method

specificity with respect to rinse solvent.

Rinse Recovery Results

(Stainless Steel Coupon)Stainless

Steel

Coupons

Sample ID

PreparationMean

Peak Area

Exp

mg/mL

Theory

mg/mL

%

Recovery

L1789 1 23.82400 0.9623

1.0268

94

L1791 2 23.09130 0.9327 91

L1792 3 23.20498 0.9373 91

Average 92

% RSD 2

Stainless

Steel

CouponsSample ID

PreparationMean

Peak Area

Exp

mg/mL

Theory

mg/mL

%

Recovery

L1789 1 227.24304 9.1784

10.0062

92

L1791 2 228.65497 9.2354 92

L1792 3 237.01920 9.5732 96

Average 93

% RSD 2


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Rinse Recovery Results(Glass Coupon)

Glass

Coupons

Sample ID

PreparationMean

Peak Area

Exp

mg/mL

Theory

mg/mL

%

Recovery

L1789 1 19.59525 0.9760

1.0083

97

L1791 2 19.22551 0.9575 95

L1792 3 19.53346 0.9729 96

Average 96

% RSD 1

Glass

Coupons

Sample ID

PreparationMean

Peak Area

Exp

mg/mL

Theory

mg/mL

%

Recovery

L1789 1 195.77969 9.7509

10.1832

96

L1791 2 194.53680 9.6890 95

L1792 3 193.89510 9.6571 95Average 95

% RSD 0

Swabs Recovery Study (Flow Diagram)

Clean Coupons

(cleaning solutionand DI water)

Spike coupon (above and

below acceptance limit)

Prepare Stock

Solution(in methanol)

Analyze samples

(dilute or concentrate asneeded)

Swab coupon

(use 2 swabs)

Extract Swabs

(shake or vortex)


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Swabbing Coupon (Stainless Steel, Glass,Teflon)

Swabs Extraction


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Swabs Recovery Study

Moisten swabs with extraction solvent and squeeze the swabs.

Swab the plate back and forth with one side and up and down with other

side.

Place the swab into a clean vial containing the required volume of

extraction solvent.

Cut off and discard the handle of the swab and vortex or shake the

solution and analyze as per defined analytical procedure.

The procedure is repeated with the second swab to ensure that no

significant amount of analyte is left on the plate.

Repeat the procedure without active residue to address the method

specificity with respect to rinse solvent and swab extractables.

TLC Method

Use series of standard dilutions to estimate the level

of residue.

Evaporate cleaning sample to dryness and re-dissolve

in method solvent.

Solvent grade test is important.

Bracket residual concentration for more accurate

visual determination.Note: Consider CAD detector if TLC is not sensitive (Use of Universal HPLC

Detection for Cleaning Validation, ESA Biosciences Application Notes)


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TLC Method Validation

Limit of detection

lowest visualized concentration

Repeatability

confirm spot intensity

confirm Rf value

Specificity

check for interferences with evaporated solvent

Rinse recovery

Swabs recovery

Conductivity Method Validation (Linearity)

Sodium Containing Compound

Concentration (g/mL) Conductivity ( S)

Blank 3.6

2.3815 2.9

4.7630 7.1

9.5260 14.4

14.2890 22.0

19.0520 29.4

Sodium Compound Linearity

y = 1.5816x - 0.6591

0

5

10

15

20

25

30

35

0 5 10 15 20

Conc. (g/mL)

Conductivity(S

)


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Conductivity Method Validation(Rinse Recovery)

Coupon Type Prep. No. % Recovery Avg. % Rec.

Stainless Steel

1 97.2

1012 99.6

3 105.7

Teflon

1 93.6

942 92.4

3 96.0

Glass

1 94.8

982 100.8

3 98.4

mg Residue = Std g/mL x 10-3mg/ g x 1000 mL/L x Total Solvent used, L

mg Residue = 5 g/mL x 10-3mg/ g x 1000 mL/L x 10L = 50 mg

Minimum batch = mg Residue/20 mg/kg

Minimum batch = 50 mg Residue/20 mg/kg = 2.5 kg

Got itbetter not


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TOC Methodology

Oxidation of carbon and detection of carbon dioxide

Oxidation techniques: photocatalytic, chemical, high-

temperature combustion

Carbon dioxide is measured by non-dispersive infrared

detector

Any remaining carbon in the sample results in TOC

TOC is classified as a non-specific method

High sensitivity (ppb range)

Detects all carbon containing compounds (active, excipients,

cleaning agents)

TOC References

How to Develop and Validate a Total Organic Carbon Method for Cleaning

Applications (Karen Clark, PDA Journal of Pharmaceutical Science and

Technology, 2001, Vol. 55, No. 5)

TOC Analysis of Compounds with Low Water Solubility; Evaluation of

Swab Recoveries for Cleaning Validation Applications(GE Water &

Process Technologies, Application Notes)

TOC Surface Swab Recovery Studies, An Integral Component of Robust

Cleaning Validation Program (Keith Bader, Hyde Engineering + Consulting,presentation 2009)

Biopharmaceutical Facility Cleaning Validation Using the Total Organic

Carbon Test (BioPharm International Magazine, 2010, Vol. 23, No. 6)


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Conclusions

Coordinate cleaning validation with production (equipment)

and R&D chemists (solubility, degradation)

Define the analyte to be tested (API/intermediate/by-product)

Define acceptance criteria (starting point for method

sensitivity)

Design analytical method for its intended use (specific/non-

specific, short, easy to operate)

Confirm the method works on selected surfaces and swabs Validate the method (method conditions, rinse and swabs

recovery)

EXPLICACIÓN DE VALIDACIÓN DE LIMPIEZA

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