Overcome Top Challenges in Analytical Method Development and Validation Q. Chan Li, Ph.D. Analytical Development US, Boehringer Ingelheim Pharmaceuticals, Inc. IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014
Outline Ask three questions before method development and validation Top challenges for method development (MD) Top challenges for method validation (MV) IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 2
Questions to ask before method development and validation What is the stage of product/clinical development (Preclinical, Phase 1, Phase 2, Phase 3, QCcommercial)? Where in the process/product development is the method used? (process monitoring, in-process testing, release testing, stability testing) What is the purpose of the method (assay, dissolution, impurity analysis, genotoxic impurity )? IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 3
Top challenges for method development (MD) How to select target analytes (impurities) for method development? Sample preparation and stability issues Method development screening (column types, mobile phases, ionic strength, ph, temperature) Chromatographic difficulties (critical pair, peak tailing ) Method conversions (UHPLC to HPLC, HPLC-UV to HPLC-MS compatible) Separation of multiple stereoisomers Quantitation limits and difficult detection IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 4
Top challenges for method development - How to select target analytes (impurities) for method development? Content uniformity, Dissolution, HPLC Assay: major component (e.g., API, DS) The API is separated from impurities. Aim for a short run HPLC impurity analysis Impurities reasonably expected based on knowledge of the process and product Work with DS / DP manufacture units to construct a list of impurities Potential process impurities, byproducts, including stereoisomers Potential degradation products Actual impurities Genotoxic impurities (GTI) Now called DNA reactive (mutagenic) impurities by ICH M7 (use GTI for convenience) GTI is separated from all other components IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 5
Top challenges for method development - How to select impurities? Case 1, Example 1: 11 potential impurities X OH OH R 1 OMe R 1 OMe + O R O 2 N N R 2 R 3 R 1 R 2 R 4 N OH OMe O + R 1 R 2 X N OH OMe O 676 677 A 676 R 1 R 2 R 3 N O O OMe R 1 R 2 R 4 N O O OMe R 1 R 2 X N O O OMe 678 B C API R 1 R 2 R 3 N 790 O O OH R 1 R 2 R 4 N G O O OH R 1 R 2 X N H O O OH R 3 R 1 OH OH R 1 R O 2 N D R 2 R 4 N E OH OH O R 1 R 2 X N F OH OH O 6
Top challenges for method development - Sample preparation and stability issues Solubility For sparingly water soluble materials, dissolve in organic solvent(s) first, then dilute with diluent (or water). Or use diluent with high organic content Sonication, shaking Excipients can make DP sample preparations more challenging Solubilization, sonication, shaking, centrifugation, filtration Insufficient materials available in early stages of development Extraction to enrich very low levels of analytes, e.g., GTI mesylates Derivatization Stability Refrigeration Solvent selection (recent case uses 80/20 methanol/water to minimize hydrolysis) Protection from heat, humidity, light Need to validate recovery (accuracy) for sample preparations IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 7
Top challenges for method development - Sample preparation and stability issues: Case 2 - Capsule case API is sparingly soluble in water Capsules are color coded Diluent: 0.1% TFA in acetonitrile/water 70/30 (v/v) Capsule solution preparation Place capsules in a flask and add diluent to 80% of the volume Ultrasonicate with periodic swirling for approximately 30 minutes Place flask on reciprocating shaker for 30 min Dilute to volume mark with diluent. Centrifuge to separate insoluble colorants from clear supernatant Pipet supernatant and dilute to desired concentration Protect sample solutions from light as much as possible during preparation IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 8
Top challenges for method development - Sample preparation: Case 1: Insufficient materials 11 potential impurities for API 790 (from Case 1) Only 4 authentic materials available Material Compound Major Impurities # 1 677 ~ 1% A ~ 2% 676 # 2 678 ~ 1% B ~ 2% C # 3 DS 790 ~ 1% G ~ 2% H # 4 (hydrolysis of 678) D ~ 1% E ~ 1% F HPLC selectivity solution 1:1:1:1 mixture of materials # 1 + 2 + 3 + 4 Inject selectivity solution, not 4 individual material solutions, for MD screening conditions After HPLC conditions are finalized, inject 4 individual material solutions Peak assignments based on retention times, relative peak intensities, and structures Peak assignments to be confirmed by LC-MS IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 9
Top challenges for method development - MD screening: Case 1 - UPLC MD screening by using Fusion AE Columns Columns 0.05% Trifloroacetic acid (TFA) in water, ph 2 20 mm Ammonium bicarbonate buffer, ph 8 Acquity UPLC HSS T3, 1.8 µm, 2.1x50 mm Acquity UPLC BEH phenyl, 1.7 µm, 2.1x50 mm Acetonitrile (ACN) Acquity UPLC BEH Shield RP18, 1.7 µm, 2.1x50 mm Methanol (MeOH) Zorbax SB-C18, 1.8 µm, 2.1x50 mm 20-100 % B in 10 min, 0.6 ml/min, 220 nm, 40 C 30-100% B in 12 min, hold 100% B for 3 min Waters UHPLC
Top challenges for method development - MD screening: Case 1- Optimal conditions from screening C18 columns with MeOH / 0.05% TFA-H 2 O
AU MD Screening - MD screening: Case 1 Convert UHPLC to HPLC Zorbax SB-C18, 2.1 x 50 um, 1.8 µm XBridge C18 column, 2.1 x 50 um, 2.5 µm XBridge C18 column, 3.0 x 100 um, 3.5 µm 0.60 0.40 Agilent 1100 XBridge C18, 3.0 x 100 mm, 3.5 µm 220 nm, 8 µl injection, 0.6 ml/min A = 0.05% TFA in water, B = MeOH GE: 40-100% B in 12 min, hold for 3 min Column 40 C 0.20 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Minutes
Top challenges for method development - MD screening: Case 1 DryLab 3-step gradient simulation XBridge C18, 3.0 x 100 mm, 3.5 µm A = 0.05%TFA-H 2 O, B = MeOH
Top challenges for method development - MD screening Case 1 : three -step gradient experiment Agilent 1100 Xbridge C18, 3.0 x 100 mm, 3.5 µm 220 nm, 8 µl injection, 0.6 ml/min A = 0.05% TFA in water, B = MeOH Column 40 C
Top challenges for MD - MD screening: Case 1 DryLab two -step gradient simulation XBridge C18, 3.0 x 100 mm, 3.5 µm; MeOH and 0.05%TFA-H 2 O 5 10 15
AU Top challenges for method development - MD screening: Case 1 two -step gradient experiment 0.06 0.04 Ignore the imp in an Impurity Agilent 1100 XBridge C18, 3.0 x 100 mm, 3.5 µm 220 nm, 8 µl injection, 0.6 ml/min A = 0.05% TFA in water, B = MeOH Column 40 C 0.02 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Minutes
Top challenges for method development Case 1: Peak ID by HPLC retention times, peak intensities, structures
Top challenges for method development Case 1: Peak ID confirmed by LC-MS (exact match) Method developed in 7 days! Final HPLC Conditions: Mobile Phase: A: 0.05% TFA in water B: methanol Column: XBridge C18, 3.0 x 100 mm, 3.5 µm Column temperature: 40 C Injection: 6 µl Flow rate: 0.6 ml/min Wavelength: 220 nm Run time: 23 min Gradient: Time (min) %B 0 50 1.0 50 3.0 65 17.0 95 18.0 100 20.0 100 18
Top challenges for MD - MD screening: Case 3 - A recent case Mobile Phases Aqueous (3): Columns Agilent Eclipse Plus C18 (2.1 x 50 mm, 1.8 µm) 0.1% TFA, ph 2 Waters Xbridge C18 (3 x 50 mm, 2.5 µm) 10 mm Ammonium Acetate, ph 4 Agilent Zorbax SB-Aq (2.1 x 50 mm, 3.5µm) 10 mm Ammonium Acetate, ph 6.8 Agilent Eclipse Plus Phenyl-Hexyl (2.1 x 50 mm, 1.8 µm) Organics (2): Acetonitrile, Methanol Waters Aquity UPLC HSS T3 (2.1 x 50 mm, 1.8 µm) Agilent Zorbax SB C8 (2.1 x 50 mm, 1.8 µm) 19
Top challenges for MD - MD screening: Case 3 - A recent case Convert from UHPLC column to HLPC column and change to phosphate buffer for late-stage development Parameter Condition Mobile Phase A 0.2 % Diammonium hydrogen phosphate ph adjusted to 7 with phosphoric acid Mobile Phase B Acetonitrile Column Agilent Zorbax SB-AQ, 3.5 µm, 3 x 100 mm Column temp 25 0 C Flow 0.6 ml/min* Gradient 0-95% B in 20 min (1 min initial hold)* Wavelength 225 nm Injection volume 5 µl* *Final method conditions: 1ml/min, 80% B final gradient, 15 µl injection 20
Top challenges for method development -MD screening: Case 3 - conversion from HPLC-UV to HPLC-MS AU AU 0.14 0.12 0.10 0.2% (NH 4 ) 2 HPO4, ph 7.0 0.2% Diammonium hydrogen phosphate, ph 7 SM 2 Imp 2 Potential Imp 0.08 0.06 SM 1 0.04 0.02 Imp 1 H2O2 deg Imp 3 0.00-0.02 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 Minutes Equivalent Selectivity 0.12 0.10 0.08 10 mm 10mM ammonium Ammonium Acetate, acetate, ph ph 6.8 6.8 SM 2 Imp 2 Potential Imp 0.06 0.04 SM 1 0.02 Imp 1 0.00 H2O2 deg Imp 3-0.02-0.04 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 Minutes 21
Top challenges for method development - Separation of multiple stereoisomers in DS Case 4 API 877 has 3 chiral centers 8 stereoisomers Two chiral precursors 818 and 121 818 has one chiral center 121 has two chiral centers Develop a control strategy as per ICH Q11 Chiral purities of both precursors are controlled Precursor 818 consistently shows enantiomer <0.05% 4 API stereoisomers (chart) Still add the stereoisomer resulting from 818 enantiomer 5 API stereoisomers Chiral HPLC method targets 5 compounds: 937, 938, 408, 409, and 877 (API) API 877 (3 chiral centers, CC) 8 isomers 818 (1 CC) Theory: 2 isomers Reality: 1 isomer 4 isomers 121 (2 CC) Theory: 4 isomers 22
Top challenges for method development - Separation of multiple stereoisomers Case 4 Variables Weak Solvent Strong Solvent Temperature Column Heptane 1-Propanol 25 C IA (4.6 x 150 mm, 3 µm) 0.1% DEA in Heptane* 2-Propanol 40 C IB (4.6 x 150 mm, 5 µm) IC (4.6 x 150 mm, 3 µm) Ethanol ID (4.6 x 150 mm, 3 µm) IF (4.6 x 150 mm, 3 µm) *For acidic samples replace diethylamine with trifluoroacetic acid Constants: Injection volume Mobile phase gradient, 20%-90% Strong solvent in 30 min, 1 ml/min Rapid Chiral HPLC Method Development Using Fusion Software 25 November 2014 23
Top challenges for method development - Separation of multiple stereoisomers Case 4 IC Column, Ethanol, 40 C The weak solvent is Heptane with 0.1% diethylamine IB Column, 2-Propanol, 25 C IC Column, 1-Propanol, 25 C 24
Top challenges for method development - Case 4 - Final chromatographic conditions Chiracel IC column, 4.6 x 150mm, 3µm A = Heptane with 0.1% diethylamine, B = 1-propanol (B) 40%-90% B in 30 min, 1.0 ml/min, 40 C 25
Top challenges for method development - Difficult detection for LC methods Analyte type Practical solutions Analytes with very different max Non-chromophoric analytes, GTI with low QL (ppm) Non-chromophoric analytes Making non-chromophoric analytes suitable for UV detection Very low levels of impurities (GTIs) Different UV wavelengths MS detector Charged aerosol detector (CAD) Evaporative light scattering detector (ELSD) Derivatization Sample enrichment (e.g., extraction, evaporation) IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 26
Top challenges for method development - Difficult detection: Case 5 - using CAD detection R 1 NH NH 2 + IN 2 (not shown) (poor chromophore) R 1 N Cl R 2 IN 1 R 1 and R 2 are aliphatic groups R 2 IN 3 N Challenges in GC FID IN 3 (parent), IN 1 and IN 2 are salts, difficult to volatilize without converting them to free bases Free base IN 1 showed poor chromatography with severe tailing; unacceptable recovery Derivatization to minimize the tailing is difficult Challenges in HPLC-UV There is NO single wavelength (e.g., 195 nm) appropriate for detecting all 3 Impurity analysis by area percent is not accurate HPLC-UV method needs individual reference materials Corona Charged Aerosol Detector (CAD) seemed to offer a better alternative 27
mv Top challenges for method development - Difficult detection: Case 5 : HPLC CAD final conditions 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00-10.00 Agilent SB-Ag, 4.6 x 150 mm, 3.5 µm CAD, pressure 3.5 psi, range 100mP Heater temp: off; sampling rate 10 15µL injection, 1.2 ml/min A = 0.1% HFBA in 98/2 (v/v) water/ MeOH; B = Acetonitrile; Column 40 C 1 23 4 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 Minutes 5 6 Selectivity solution containing 100% APISM, 0.5% of IN 1 and IN 2 7 8 9 10 11 12 13 Peak Peak Name Number 1 IN1 2 RRT 0.54 3 RRT 0.55 4 RRT 0.59 5 IN3 6 RRT 1.04 7 IN2 8 RRT 1.22 9 RRT 1.31 10 RRT 1.39 11 RRT 1.43 12 Background Peak 13 RRT 1.50 Have successfully used HPLC-CAD to analyze intermediates, amino acids, counter ions, co-crystal components. 28
Top challenges for method development - Difficult detection: Case 6: using derivatization A limit test for formaldehyde in a BI DS (J. Chrom. Sci. 2008, 46 (6), pp. 461-465) Derivatization: HCHO + 2,4-dinitrophenylhydrazine (DNPH) Schiff base Choose a well established chemical reaction mau 900 800 700 600 500 400 *DAD1, 3.710 (838 mau, - ) Ref=2.924 & 4.510 of 41018012.D DNPH ACE C8, 150 mm x 4.6 mm, 3 um 45:55 acetonitrile /water (v/v) 1.0 ml/min, = 360 nm 300 200 220 240 260 280 300 320 340 360 380 nm mau 80 70 *DAD1, 6.179 (72.1 mau,apx) Ref=6.539 of 41018004.D HCHO-DNPH 60 50 40 30 20 10 220 240 260 280 300 320 340 360 380 *DAD1, 7.757 (350 mau, - ) Ref=7.597 & 7.811 of SIG10002.D nm QL =0.3 ppm F E D C B A mau 300 250 200 BI DS 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 RT (min): DNPH, 3.82; HCHO-DNPH, 6.37; BI DS, 8.14 150 100 50 0 220 240 260 280 300 320 340 360 380 IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 nm A. Mobile Phase (Blank); B. HCHO (Blank); C. DNPH; D. BI DS; E. Derivatization standard at QL 0.3 ppm F. Derivatization sample (spiked with formaldehyde, ~3.33 ppm) 29
Top challenges for method validation (MV) Guidances How much validation needs to be done? Japanese regulatory Authorities (MHLW) s special requirement for intermediate precision Bias in linearity Mass imbalance Recovery out of range Robustness issues (Workshop S4) Method verification (Workshop S11) Revalidation (Workshop S14) IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 30
Top challenges for method validation - Guidances and recent references ICH Q2(R1): Validation of Analytical Procedures: Text and Methodology (Step 4, November 2005) FDA Reviewer Guidance on Validation of Chromatographic Methods (November 1994) USP/NF General Chapter <621> Chromatography General Chapter <1224> Transfer of Analytical Procedures General Chapter <1225> Validation of Compendial Procedures General Chapter <1226> Verification of Compendial Procedures Lifecycle approach (recent) D. Chambers et al., GMPs for Method Validation in Early Development: An Industry Perspective (Part II), Pharm. Technol. 36 (7) 76 84 (2012) FDA Draft Guidance for Industry: Analytical Procedures and Methods Validation for Drugs and Biologics, February 2014 CMC USP Validation and Verification Expert Panel on Lifecycle Management of Analytical Procedures: Method Development, Procedure Performance, Pharmacopeial Forum(PF) 39(6) IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 31
Top challenges for method validation - How much validation needs to be done? ICH Q2, FDA review guidance, and USP general chapters are for phase 3 and registration for NDA, ANDAs, BLAs, and DMFs For Phase 3 and registration, intermediate precision validation design that also meets Japanese Regulatory Authorities (MHLW)) s special requirement Day Analyst Apparatus Column Preparation 1 A A A 2 2 A B A 2 3 A A B 2 4 (1) B B A 2 5 (2) B A B 2 6 (3) B B B 2 Staged approach to clinical phases 1-2 Do not validate intermediate precision and robustness (except solution stability) Select fewer impurities for linearity, precision, accuracy validation Scale back extent of accuracy validation, e.g., 2 x 2 instead of 3 x 3 IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 32
Top challenges for method validation - How much validation needs to be done? Recent, broad thinking of lifecycle approach - FDA draft guidance on MV (2014) Revalidation, alternative method, method comparability study, method transfer Workshops S4, S11, S14 Method qualification (or qualified methods) IQ paper (2012) Scientific sound, reliable methods used for characterization work, such as reference standards, IPT, GLP analysis, the scientific prediction of shelf-life Not used for GMP release of clinical materials Method verification USP general chapter <1226> Assessing the suitability of a compendial test procedure under the conditions of actual use Verification is not required for basic compendial test procedures, such as LOD, ROI, various wet chemical procedures (e.g., acid value), ph measurements. BUT consideration is given to any new or different sample handling or solution preparation requirements IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 33
Top challenges for method validation (MV) Bias in linearity Bias is the intercept on the Y-axis (response like peak area) Co-elution of components with very different responses at a given Purity of the analyte is not accurately determined Background interference When assay and impurity are in one method Prefer to use the assay standard response for calculating assay and impurities When the assay standard response is not linear from 0.05% (QL) - 120%, then an impurity standard (e.g., at 0.5%) needs to be used IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 34
Top challenges for method validation (MV) Mass imbalance (desired to have assay + impurities within 97-100%) Solution stress Degradation products with very different UV responses, mass balance is often outside 97-100% Solutions: generate 5-20% degradation; set 95-100% for early stage development? However, outside 97-100% might indicate a real problem with the material (e.g., degradation, inorganic, volatile impurities) We encountered problems with degradation and inorganic impurities, which were revealed by low assays Recovery out of range Sample preparation (solubility, dilution ) Peak contamination from dirty glassware, mobile phase, column, or system... Matrix effect - any change in manufacture and quality of excipients, filter We experienced such changes (suppliers did not notify) and did time-consuming investigations Degradation IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 35
Acknowledgments Lily Du Jessica Vazquez Oliver Taylor (2014 summer intern from New York University) Natalia Luneva Shaun Mendonsa Ken Cohen IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 36
Backup IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 37
Principles of CAD http://coronacad.com/cad_overview.htm Nebulization Evaporation Particle Monitoring Particle Charging 38
Corona Charged Aerosol Detector (CAD) No analyte is actually ionized by CAD CAD is a Quadratic Response Detector Linear-like over short ranges and low concentrations Signal increases with increased organic content Basic ph mobile phase will result in low S/N Tetrahydrofuran, hexanes and halogenated solvents like dichloromethane, and chloroform may require heated nebulization Corona Plus 39
HPLC Method Translation (Conversion) Ref: Ronald E. Majors, Method Translation in Liquid Chromatography, LCGC IVT Analytical Method Validation, San Diego, CA, 2-4 December 2014 40
Non-Volatile to Volatile Buffer Conversion for LC-MS Structural Identification Replace phosphate with volatile buffer from list below based on ph Use the same column, temp, organic, gradient, flow rate, etc. Perform minor fine tuning as needed to get equivalent selectivity Gradient shape, % B, flow rate, temp, etc. Phosphate Buffer ph Volatile Buffer 2.0 TFA 3.0 Formic Acid 4.0 Acetic Acid 5.0 Ammonium Acetate (acetate) 6.0 Ammonium Bicarbonate (carbonate, 1) 7.0 Ammonium Acetate (ammonium) 8.0 Ammonium Hydroxide 9.0 Ammonium Bicarbonate (ammonium) 41
Top challenges for method validation - How much MV needs to be done? IQ paper recommendations 42