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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 for analytical 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 <645> ( 10 S) pH test USP <791> (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 UVmax 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 a gray 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/mL) were not Included in Chart 40 Solubility in Methanol (mg/mL)

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

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25

20

15

10

0 GMP Compounds

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Maximum Allowable Carryover (MAC) and Acceptance Limits

RINSE SWAB Residue limit is 0.002% => Residue Limit = 20 mg 20 mg for 1kg batch Limit for swab = Typical final rinse is 10L => (MAC/Total Surface Area x Swab 0.002 mg/mL residue Area)/ Volume of Swab solution concentration Example: (20mg/40000cm2 x Limit for rinse = 0.002 100 cm2)/ 10 mL = 0.005 mg/mL 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 Selection

Compound Structure Method Validation Analytical Validation Rinse/Swab Recovery Fentanyl
N N O

HPLC-UV

CH 3

OH

Analytical Validation Rinse/Swab Recovery TLC

Oxandrolone
O

Inorganic Product

Sodium Containing Compound

Conductivity

Analytical Validation Rinse/Swab Recovery

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 Defining a 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
mAU 30

Std at 206, 220, 254 nm

DAD1 A, Sig=206,4 Ref=360,100 (CLEANING\10062107.D) DAD1 B, Sig=220,4 Ref=360,100 (CLEANING\10062107.D) DAD1 C, Sig=254,4 Ref =360,100 (CLEANING\10062107.D)

25

20

15

5 .3 5 8

10

5 5 .3 5 8

200

210

220

230

240

250

260

270

280

290

nm

5 .3 5 8

-5

min

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Solvent Grade
Methanol ACS Grade
DAD1 A, Sig=206,4 Ref =360,100 (CLEANING\10062108.D) DAD1 B, Sig=220,4 Ref =360,100 (CLEANING\10062108.D) DAD1 C, Sig=254,4 Ref=360,100 (CLEANING\10062108.D) mAU m AU

Methanol Tech Grade

DAD1 A, Sig=206,4 Ref=360,100 (CLEANING\10062109.D) DAD1 B, Sig=220,4 Ref=360,100 (CLEANING\10062109.D) DAD1 C, Sig=254,4 Ref =360,100 (CLEANING\10062109.D)

30

30

20 20

10 10

0 0

5 .2 0 9

-10

-10 0 1 2 3 4 5 6 7 8 9 m in 0 1 2 3 4 5 6 7 8 9 min

Diluent (Split Peak)

100% Methanol
VWD1 A, Wavelength=246 nm (30202\10051201.D) mAU 400 350 300 250 200 150 100 50 0 0 0 1 2 3 4 5 6 m 0 1 2 3 4 5 6 m 20 3.428 - Analyte 60 mAU 80 3.425 - Analyte

50% Methanol
VWD1 A, Wavelength=246 nm (30202\10051202.D)

40

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Swabs Extractables
Swab Recovery
VWD1 A, Wavelength=242 nm (30202\09121524.D) mAU 35 30 25 20 15 10 5 0 -5 -10 0

Blank Swab Extracted (2 inj)

VWD1 A, Wavelength=242 nm (30202\09121508.D) mAU 35 30 25 20 15 10 5 0 -5 -10 0 VWD1 A, Wavelength=242 nm (30202\09121525.D) mAU 35 30 25 20 15 10 5 0 -5 -10 0 6.495 - Swab extractable 1 2 3 4 5 6 7

5.189 - Analyte 6

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 350.00000 R2 = 0.9999 300.00000 250.00000 area 200.00000 150.00000 100.00000 50.00000 0.00000 0.0000

2.0000

4.0000

6.0000 ug/ml

8.0000

10.0000 12.0000

Method Validation (Accuracy/Precision)

Sample ID Injection 1 1 g/mL 2 3 1 5 g/mL 2 3 1 10 g/mL 2 3 Theory mg/mL 0.00109 0.00109 0.00109 0.00543 0.00543 0.00543 0.01085 0.01085 0.01085 Peak Area 33.47281 33.18301 33.26467 163.56015 163.24034 163.38344 330.45963 329.64816 330.35934 Exp. mg/mL 0.001 0.001 0.001 0.005 0.005 0.005 0.011 0.011 0.011 % Recovery 102.629 101.334 101.584 99.896 99.701 99.788 100.916 100.668 100.885 100.822 0.957 0.949 Std. Deviation9 % RSD9 100.823 99.795 101.849 % Recovery Averages

Average (1, 5, 10 g/mL)

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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 Summary

Validation Parameters System Suitability Test Parameters % RSD6 working standard Correlation coefficient (r) Method Linearity (1 to 10 g/mL) y-Intercept Slope Residual standard deviation % Recovery % RSD9 Blank injections S/N at 1 g/mL Concentration at S/N = 3 Limits 5% 0.995 Informational Informational Informational 80 120% 10% No interfering peaks 10 Report g/mL Results 0.2% 1.000 -0.01227 30.36423 0.86422 101% 1% No interfering peaks 22 0.15

Method Accuracy (1, 5, 10 g/mL) Method Precision (1, 5, 10 g/mL) Specificity Method Quantitation Limit Method Detection Limit

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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 L1789 L1791 L1792 Preparation 1 2 3 Mean Peak Area 23.82400 23.09130 23.20498 Exp mg/mL 0.9623 0.9327 0.9373 Theory mg/mL 1.0268 Average % RSD % Recovery 94 91 91 92 2

Stainless Steel Coupons Sample ID L1789 L1791 L1792

Preparation 1 2 3

Mean Peak Area 227.24304 228.65497 237.01920

Exp mg/mL 9.1784 9.2354 9.5732

Theory mg/mL 10.0062 Average % RSD

% Recovery 92 92 96 93 2

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Rinse Recovery Results (Glass Coupon)

Glass Coupons Sample ID L1789 L1791 L1792 Preparation 1 2 3 Mean Peak Area 19.59525 19.22551 19.53346 Exp mg/mL 0.9760 0.9575 0.9729 Theory mg/mL 1.0083 Average % RSD % Recovery 97 95 96 96 1

Glass Coupons Sample ID L1789 L1791 L1792

Preparation 1 2 3

Mean Peak Area 195.77969 194.53680 193.89510

Exp mg/mL 9.7509 9.6890 9.6571

Theory mg/mL 10.1832 Average % RSD

% Recovery 96 95 95 95 0

Swabs Recovery Study (Flow Diagram)

Clean Coupons (cleaning solution and DI water) Prepare Stock Solution (in methanol)

Spike coupon (above and below acceptance limit)

Swab coupon (use 2 swabs)

Extract Swabs (shake or vortex)

Analyze samples (dilute or concentrate as needed)

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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 35 30 25 20 15 10 5 0 0 5 10
Conc. (g/mL)

Conductivity (S)

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Conductivity Method Validation (Rinse Recovery)

Coupon Type Stainless Steel Teflon Glass Prep. No. 1 2 3 1 2 3 1 2 3 % Recovery 97.2 99.6 105.7 93.6 92.4 96.0 94.8 100.8 98.4 Avg. % Rec. 101 94 98

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, hightemperature 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/nonspecific, short, easy to operate) Confirm the method works on selected surfaces and swabs Validate the method (method conditions, rinse and swabs recovery)

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