explicaciÓn de validaciÓn de limpieza
TRANSCRIPT
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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
Analytical Validation
Rinse/Swab Recovery
Inorganic Product
Sodium Containing
Compound Conductivity
Analytical Validation
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
VWD1 A, Wavelength=246 nm (30202\10051202.D)
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
VWD1 A, Wavelength=242 nm (30202\09121524.D)
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
VWD1 A, Wavelength=242 nm (30202\09121508.D)
m0 1 2 3 4 5 6 7 8 9
mAU
-10
-5
0
5
10
15
20
25
30
35
VWD1 A, Wavelength=242 nm (30202\09121525.D)
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)