Tides 2007 Conference Las Vegas, May 23, 2007 UPLC for Synthetic Peptides: A User`s Perspective Holger Hermann, Jens Donath* Lonza AG, Switzerland
Welcome to Lonza! One of the leading CMO Production facilities worldwide Europe USA Asia Switzerland Belgium Czech Republic UK Spain China Singapore slide 2
QC Tasks at Lonza Visp Method Development Method Validation & Transfer Support of R&D Activities Structure elucidation Raw material Testing In-Process Testing (24/7) Release Testing of finished Products Cleaning (Swab / Rinse) Stability Testing slide 3
Options for Speed Other techniques (NMR, etc) New instruments/columns UPLC / <2μm types Method improvements Combination of all PAT / Online analytics Optimize workflow More resources (FTE, instruments) slide 4
Why UPLC? Use of smaller particles (< 2 µm) + speed up of analysis (higher flow rates) + higher sensitivity (less peak broadening) + better resolution (more theoretical plates/length) - higher back pressure (due to smaller particles) slide 5
Need for Speed Peptide synthesis Crude protected peptide Deprotection of peptide Purified peptide Purification of peptide Crude unprotected peptide more than once Decision on OK pool Fraction control Preparative HPLC slide 6
UPLC Fraction Control 1 8-mer 0.070 0.060 0.050 0.040 0.030 0.020 UPLC 2 times faster runtime 3-4 x higher intensity better resolution 1.566 0.3 % impurity 99.0 % pure 17.403 17.857 18.393 UPLC method runtime 32 min. flow : 0.25 ml / min. column : Waters Acquity BEH Shield 0.3 % impurity 22.092 22.437 RP18, 2.1x100mm, 1.7µm 25.154 25.351 25.959 26.138 26.495 0.000 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 26.00 28.00 30.00 32.00 0.020 0.018 0.016 0.014 0.012 0.008 0.006 0.004 2.466 1 0.3 % impurity 98.9 % pure 20.086 20.824 21.442 21.875 2 29.613 3 37.254 4 5 41.752 42.118 HPLC method runtime 68 min. flow : 1 ml / min. column: Waters XBridge Shield RP18, 4.6x150mm, 3.5µm 0.002 0.000 0.2 % impurity -0.002 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 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 58.00 60.00 62.00 64.00 66.00 68.00 slide 7
UPLC Fraction Control 1 8-mer 0.034 0.032 0.030 0.028 0.026 0.024 0.022 0.020 0.018 UPLC 2 times faster runtime 3-4 x higher intensity better resolution 0.016 0.014 0.012 0.008 15.625 16.159 18.520 18.653 19.186 20.870 21.455 21.634 21.783 22.773 23.403 23.633 23.780 19.650 22.157 24.325 24.563 24.690 24.750 20.406 24.121 17.863 22.485 22.536 22.995 24.468 0.006 0.004 0.002 0.000 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 0.035 0.030 0.025 0.020 0.015 0.005 0.000-0.005 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.00 35.00 36.00 37.00 38.00 39.00 40.00 slide 8 17.475 17.641 22.765 23.221 23.757 24.472 26.164 26.802 28.672 30.649 35.690 36.515 36.920 37.973 38.210 39.705 29.797 31.246 20.813 37.502 HPLC
UPLC Fraction Control 2 Cyclic 9-mer 0.024 0.022 0.020 0.018 0.016 0.014 0.012 HPLC SFI-323-PG1-33.112 52.476 HPLC Release Method Phenomenex Synergi MAX-RP C18 250x4.6mm, 4mm 0.008 13.356 50.825 0.006 0.004 0.002 12.674 14.601 15.758 17.982 18.894 20.759 26.421 27.558 28.205 29.793 30.907 31.595 35.900 39.415 41.070 42.090 46.529 48.911 49.900 51.900 52.967 53.325 54.099 54.662 0.000-0.002-0.004 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 Run time >60min Resolution of critical impurities needed Column not UPLC compatible Solution for IPC 2 separate methods, dedicated instruments slide 9
UPLC Fraction Control 2 Cyclic 9-mer 0.050 0.048 0.046 0.044 0.042 0.040 0.038 0.036 HPLC MP-003-22.066 23.838 IPC A Waters Symmetry Shield RP8 250x4.6mm, 5μm 0.034 0.032 0.030 0.028 21.269 22.976 24.111 0.026 0.024 0.022 0.020 0.018 17.812 21.736 22.493 22.713 23.449 24.324 24.886 26.162 0.016 0.014 0.012 0.008 0.006 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00 0.060 0.055 0.050 0.045 HPLC SFI-323-TG1-16.491 IPC B Agilent Zorbax C8 150x4.6mm, 3.5μm 0.040 0.035 18.287 0.030 17.951 0.025 0.020 14.620 17.422 0.015 14.197 14.302 15.109 15.321 15.511 15.722 15.833 16.185 16.911 17.034 17.235 17.714 18.598 18.721 18.990 19.311 19.397 19.508 19.886 19.959 20.385 0.005 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 slide 10
Speed and Resolution! 0.024 0.022 0.020 0.018 HPLC 0.016 0.014 0.012 0.008 0.006 0.004 0.002 0.000-0.002-0.004 0.015 0.014 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 0.013 0.012 0.011 0.009 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0.000-0.001 3.00 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 18.00 HPLC Release Method Phenomenex Synergi MAX-RP C18 250x4.6mm, 4mm UPLC IPC Method Waters BEH C18 100x2.1mm, 1.7mm same to better resolution than release 3 x faster slide 11 5.010 5.253 6.972 7.867 10.270 4.124 14.153 16.250 4.375 15.802 16.667 16.710 17.383 17.315 17.493 17.632 17.843 16.925 17.133 13.246 UPLC 12.674 15.758 14.601 17.982 18.894 20.759 26.421 27.558 28.205 29.793 30.907 31.595 35.900 39.415 41.070 46.529 42.090 48.911 49.900 51.900 52.967 53.325 54.099 54.662 13.356 50.825 52.476 SFI-323-PG1-33.112
Need for Resolution Peptide synthesis Crude protected peptide Deprotection of peptide Purified peptide Purification of peptide Crude unprotected peptide Impurity Profile slide 12
Impurity Profile I 20mer LC / MS (TOF) experiments to analyze for co-eluting compounds M 7 M 9 M 1 M 2 M 3 M 4 M 5 M 6 M 10 M 11 M 8 M 12 slide 13
Impurity Profile II A/D Converter Channel 1 from 11433_01.wiff Intensity, % 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 wrong isomer e.g. D-isomer M 8 -Tyr M W target peptide M 7 M 2 -Pro M 1 +Gly / -Leu/Ile starting material M 3 M 4 +Tyr 2.7 2.6 deletion 2.5 2.4 2.3 2.2 M 6 + Leu M 5 +Gly double hit 2.1 2.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0 Time, min slide 14
Results HPLC for Column I TM-001 0.015 0.014 0.013 0.012 0.011 0.009 0.008 0.007 0.006 0.005 target peptide TM-001_+2Pro impurity 4 TM-001_2Gly impurity 5 TM-001_+Pro TM-001_6Gly TM-001_+Tyr impurity 2 TM-001_3Gly impurity 3 impurity 1 impurity 6 0.004 0.003 0.002 0.001 0.000-0.001-0.002-0.003 15.20 15.40 15.60 15.80 16.00 16.20 16.40 16.60 16.80 17.00 17.20 17.40 17.60 17.80 18.00 18.20 18.40 18.60 slide 15
Results HPLC for Column II 0.015 0.014 0.013 0.012 0.011 0.009 0.008 TM-001_+2Pro TM-001_+Pro TM-001_6Gly TM-001_2Gly TM-001_+Tyr impurity 1 impurity 2 TM-001_3Gly TM-001 0.007 0.006 0.005 target peptide impurity 4 impurity 3 impurity 5 impurity 6 0.004 0.003 0.002 0.001 0.000-0.001-0.002-0.003 10.00 10.20 10.40 10.60 10.80 11.00 11.20 11.40 11.60 11.80 12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40 13.60 13.80 14.00 slide 16
HPLC Method Development Column Screening Waters XTerra, XBridge (C18) Jupiter Proteo (C12) Synergi (C18) Discovery HS (C18), RP-Amide (C16) SunFire C8, Sunfire 18 Kromasil C4 ZirChrom Carb (polare phase) Lichrospher NH 2 YMC ODS AQ C18 anion / cation exchange columns etc. Different buffer systems ammonium acetate phosphate buffer perchlorate buffer ammonium bicarbonate buffer Different ph ph 2, ph 7, ph 10 Different organic solvents acetonitrile methanol slide 17
HPLC / Optimized 0.072 0.070 0.068 0.066 0.064 0.062 0.060 0.058 0.056 0.054 0.052 0.050 0.048 0.046 0.044 0.042 0.040 0.038 0.036 0.034 0.032 0.030 0.028 0.026 0.024 0.022 0.020 0.018 0.016 0.014 0.012 18.167 18.853 19.083 19.417 19.961 20.212 20.912 impurity 1 21.433 target peptide impurity 2 22.382 22.873 23.328 impurity 6 impurity 7 impurity 8 impurity 3 24.233 flow 1.0 ml / min. sodium acetate RP C18 300 Å 250 x 4.6mm, 5 μm 24.677 impurity 4 25.840 26.463 impurity 5 26.999 8 minutes 5 impurities 27.882 0.008 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 slide 18
UPLC / Optimized 0.015 0.014 0.013 0.012 0.011 0.009 impurity 1 19.598 20.499 target peptide impurity 3 21.118 flow 0.25 ml / min. perchlorate buffer Acquity UPLC BEH C18 150 x 2.1mm, 1.7 μm 0.008 0.007 0.006 0.005 0.004 impurity 2 20.019 impurity 4 21.544 impurity 5 21.813 impurity 6 22.048 imprity 7 22.347 impurity 8 23.214 0.003 0.002 0.001 19.143 19.826 20.282 22.728 4 minutes 8 impurities 0.000-0.001-0.002 18.00 18.20 18.40 18.60 18.80 19.00 19.20 19.40 19.60 19.80 20.00 20.20 20.40 20.60 20.80 21.00 21.20 21.40 21.60 21.80 22.00 22.20 22.40 22.60 22.80 23.00 23.20 23.40 23.60 23.80 24.00 24.20 24.40 slide 19
Transfer HPLC UPLC: Tools Waters UPLC calculator (http://www.waters.com/calc) HPLC Calculator v1.0 Developed by J.-L. VEUTHEY, D. GUILLARME, Laboratory of Analytical Pharmaceutical Chemistry, University of Geneva (http://www.unige.ch/sciences/pharm/fanal/divers/downloads.php) slide 20
HPLC Calculator v1.0 slide 21
Transfer HPLC UPLC: Our learnings Easy to transfer for small molecules (less impurities, therefore resolution usually less critical) Easy to transfer if same column type is used Otherwise: Peak assignment might become an issue (impurity markers/ms detection advantageous) More resolution potentially more peaks Transfer tools are helpful, but do not replace experts QA accepted transfer concept slide 22
UPLC Small Molecules - Pseudoproline 0.030 0.025 0.020 0.015 FMOC-Ala-Thr(Ψ MeMe pro)-oh 9.992 98.96 area % HPLC Waters XBridge C18 5.0µm, 4.6x150 mm) Inj. vol. 10 μl flow 1.0 ml/min. 0.005 0.43 5.018 0.04 6.725 0.17 7.460 7.657 0.08 0.09 9.531 0.22 12.868 0.000-0.005 0.00 1.00 2.00 3.00 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 18.00 19.00 20.00 0.030 0.025 0.020 0.015 0.005 0.000 shorter run time better resolution easy transfer 0.43 1.302 0.02 1.786 0.15 2.001 2.170 0.10 0.08 2.578 2.926 99.01 area % 0.21 4.000 UPLC Acquity UPLC BEH C18 1.7µm, 2.1x100 mm Inj. vol. 1.4 μl flow 0.61 ml/min. -0.005-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 slide 23
HPLC/UPLC - Adjustment or Change? Adjustment no re-validation proven by robustness studies Guidance: FDA ORA Laboratory Procedure #ORA-LAB.5.4.5. Revised 09/09/2005 USP Guidance drafted (e.g. Pharmacopeial Forum 31(6), 1681, 2005) Change re-validation not proven by robustness study FDA Drafted Guidance Mostly Draft Guidance, therefore to be discussed with QA Literature: M. Swartz, I. Krull, LCGC, 24(8), 770 (2006) slide 24
Adjustments Parameter ph Buffer Salt Concentration Ratio of components in the mobile phase UV Detector Wavelength Column Length Column Inner diameter Flow Rate Injector volume Particle Size Column Temperature max. Variance ± 0.2 units ±10% Components specified at 50% or less: ±30% or ±2% absolute No Deviations ±70% ±50% (ORA) ±50% Reduced as far as consistent with accepted precision and detection limits Reduced by as much as 50% ±20% - gradient adjustments? - column chemistry? - compensation for gradient delay times? slide 25
Changes Comparability Protocol The comparability protocol would be designed to demonstrate that the proposed changes in the analytical procedures improve or do not significantly change characteristics used in methods validation that are relevant to the type of analytical procedure (e.g., accuracy, precision, specificity, detection limit, quantitation limit, linearity, range) Reference: Guidance for Industry: Comparability Protocols Chemistry, Manufacturing, and Controls Information (http://www.fda.gov/cder/gmp/5427dft.pdf) slide 26
Summary UPLC powerful tool to save time in IPC. UPLC powerful tool to gain resolution for process development/improvement. Transferring HPLC to UPLC methods easy and straight forward if the same column type is used. Transferring HPLC to UPLC methods easy and straight forward for small molecules. Transferring HPLC to UPLC methods for synthetic peptides sometimes rises addition questions (peak assignment, coeluting peaks). MS detection very helpful. slide 27
Thank you for your attention!