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 1
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 2
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%. Swab sampling Sampling Procedures 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 3
Rinse sampling Sampling Procedures Covers the entire surface area Ensure chosen solvent has appropriate recovery Easier to sample Reduced number of samples Needle in the Hay? 4
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. 5
Solvent Selection DI water Remove cleaning agent Remove water soluble salts Conductivity test, USP <645> ( 10µ 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 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 μm syringe filter, dilute and compare to a standard solution of known concentration (0.1-1.0 mg/ml). 6
Solubility in Methanol (cont d) 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 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 Solubility in Methanol (mg/ml) 35 30 25 20 15 10 5 0 GMP Compounds 7
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 = 0.002 mg/ml SWAB Residue Limit = 20 mg Limit for swab = (MAC/Total Surface Area x Swab Area)/ Volume of Swab solution Example: (20mg/40000cm 2 x 100 cm 2 )/ 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 8
Method and Detection Selection Compound Structure Method Validation Fentanyl N N O HPLC-UV Analytical Validation Rinse/Swab Recovery CH 3 Oxandrolone O OH TLC Analytical Validation Rinse/Swab Recovery O Inorganic Product H 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% HClO 4 : 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 9
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 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) mau 30 25 20 15 5.358 10 5 5.358 200 210 220 230 240 250 260 270 280 290 nm 0 5.358-5 1 2 3 4 5 6 7 8 9 min 10
Solvent Grade Methanol ACS Grade DAD1 A, Sig=206, 4 Ref =360, 100 (CLEANI NG\10062108. D) DAD1 B, Sig=220, 4 Ref =360, 100 (CLEANI NG\10062108. D) DAD1 C, Sig=254, 4 Ref =360,100 (CLEANI NG\ 10062108. D) Methanol Tech Grade DAD1 A, Sig=206,4 Ref =360,100 (CLEANI NG\10062109.D) DAD1 B, Sig=220,4 Ref =360,100 (CLEANI NG\10062109.D) DAD1 C, Sig=254,4 Ref =360,100 (CLEANI NG\ 10062109. D) mau mau 30 30 20 20 10 10 0 0 5.209-10 -10 0 1 2 3 4 5 6 7 8 9 min 0 1 2 3 4 5 6 7 8 9 min Diluent (Split Peak) 100% Methanol 50% Methanol mau VWD1 A, Wavelength=246 nm (30202\10051201.D) mau VWD1 A, Wavelength=246 nm (30202\10051202.D) 400 80 350 300 250 200 3.428 - Analyte 60 40 3.425 - Analyte 150 100 20 50 0 0 0 1 2 3 4 5 6 m 0 1 2 3 4 5 6 m 11
Swabs Extractables mau Swab Recovery VWD1 A, Wavelength=242 nm (30202\09121524.D) Blank Swab Extracted (2 inj) VWD1 A, Wavelength=242 nm (30202\09121508.D) VWD1 A, Wavelength=242 nm (30202\09121525.D) mau mau 35 35 35 30 30 30 25 20 15 10 5 5.189 - Analyte 25 20 15 10 5 25 20 15 10 5 6.495 - Swab extractable 0 0 0-5 -5-5 -10 0 1 2 3 4 5 6 7 8 9 m -10 0 1 2 3 4 5 6 7 8 9 m -10 0 1 2 3 4 5 6 7 8 9 m 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 12
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 μg/ml. The five-level calibration curve was found to be linear with correlation coefficient (r) of 0.995. area Fentanyl Linearity 350.00000 R 2 = 0.9999 300.00000 250.00000 200.00000 150.00000 100.00000 50.00000 0.00000 0.0000 2.0000 4.0000 6.0000 8.0000 10.0000 12.0000 ug/ml Method Validation (Accuracy/Precision) Sample ID Injection Theory mg/ml Peak Area Exp. mg/ml % Recovery % Recovery Averages 1 μg/ml 5 μg/ml 10 μg/ml 1 0.00109 33.47281 0.001 102.629 2 0.00109 33.18301 0.001 101.334 101.849 3 0.00109 33.26467 0.001 101.584 1 0.00543 163.56015 0.005 99.896 2 0.00543 163.24034 0.005 99.701 99.795 3 0.00543 163.38344 0.005 99.788 1 0.01085 330.45963 0.011 100.916 2 0.01085 329.64816 0.011 100.668 100.823 3 0.01085 330.35934 0.011 100.885 Average (1, 5, 10 μg/ml) 100.822 Std. Deviation 9 0.957 % RSD 9 0.949 13
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 μg/ml LOQ concentration was determined. The detection limit was calculated using the determined S/N at 1 μg/ml as follows: LOD, µg/ml = Conc. of Analyte, µg/ml x 3 S/N Method Validation Summary Validation Parameters Test Parameters Limits Results System Suitability % RSD 6 working standard 5% 0.2% Correlation coefficient (r) 0.995 1.000 Method Linearity (1 to 10 µg/ml) Method Accuracy (1, 5, 10 µg/ml) Method Precision (1, 5, 10 µg/ml) y-intercept Informational -0.01227 Slope Informational 30.36423 Residual standard deviation Informational 0.86422 % Recovery 80 120% 101% % RSD 9 10% 1% Specificity Blank injections No interfering peaks No interfering peaks Method Quantitation Limit S/N at 1 µg/ml 10 22 Method Detection Limit Concentration at S/N = 3 Report µg/ml 0.15 14
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 15
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 Preparation Mean Peak Area Exp mg/ml Theory mg/ml % Recovery L1789 1 23.82400 0.9623 94 L1791 2 23.09130 0.9327 1.0268 91 L1792 3 23.20498 0.9373 91 Average 92 % RSD 2 Stainless Steel Coupons Sample ID Preparation Mean Peak Area Exp mg/ml Theory mg/ml % Recovery L1789 1 227.24304 9.1784 92 L1791 2 228.65497 9.2354 10.0062 92 L1792 3 237.01920 9.5732 96 Average 93 % RSD 2 16
Rinse Recovery Results (Glass Coupon) Glass Coupons Sample ID Preparation Mean Peak Area Exp mg/ml Theory mg/ml % Recovery L1789 1 19.59525 0.9760 97 L1791 2 19.22551 0.9575 1.0083 95 L1792 3 19.53346 0.9729 96 Average 96 % RSD 1 Glass Coupons Sample ID Preparation Mean Peak Area Exp mg/ml Theory mg/ml % Recovery L1789 1 195.77969 9.7509 96 L1791 2 194.53680 9.6890 10.1832 95 L1792 3 193.89510 9.6571 95 Average 95 % RSD 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) 17
Swabbing Coupon (Stainless Steel, Glass, Teflon) Swabs Extraction 18
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) 19
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 (µg/ml) Conductivity (µs) Blank 3.6 2.3815 2.9 4.7630 7.1 9.5260 14.4 14.2890 22.0 19.0520 29.4 Conductivity (µs) 35 30 25 20 15 10 5 0 Sodium Compound Linearity y = 1.5816x - 0.6591 0 5 10 15 20 Conc. (µg/ml) 20
Conductivity Method Validation (Rinse Recovery) Coupon Type Prep. No. % Recovery Avg. % Rec. 1 97.2 Stainless Steel 2 99.6 101 3 105.7 1 93.6 Teflon 2 92.4 94 3 96.0 1 94.8 Glass 2 100.8 98 3 98.4 mg Residue = Std µg/ml x 10-3 mg/ µg x 1000 ml/l x Total Solvent used, L mg Residue = 5 µg/ml x 10-3 mg/ µg 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 it better not 21
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) 22
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) 23