Ultra Performance Liquid Chromatography Stephan Koza, Paula Hong, and Kenneth J. Fountain Waters Corporation, 34 Maple Street, Milford, MA, USA & SEC 2010 2 8 5 2030 4 3 8 1982 2009 131 USP EPHMW USP34 3134 EP Volume 5 1800 HPLC SEC HMW USP L20 3-10 μm 7.8 mm x 30 cm EP R 12-12.5 nm 5-10 μm 7.5 mm 30 cm 125 2 μm BEH Waters Ultra Performance Liquid Chromatography UPLC HMW ACQUITY UPLC H-Class Bio ACQUITY UPLC BEH125 SEC, 1.7 μm Empower TM 3 UPLC USP EP 1
LC ACQUITY UPLC H-Class Bio TUV Sigma, 12643 4.0 mg/ml 0.01 N 276 nm 25 10 10 μl 0.4 ml/min L - 1.0 g/l / 99% / 65/15/20 v/v/v 10% 10% 0.01 N ACQUITY UPLC BEH125 SEC, 1.7 μm, 4.6 x 150 mm P/N 186006505 ACQUITY UPLC BEH125 SEC, 1.7 μm, 4.6 x 300 mm P/N 186006506 ACQUITY UPLC BEH200 SEC, 1.7 μm, 4.6 x 300 mm P/N 186005226 BioSuite TM 125 UHR SEC, 4 μm, 4.6 x 300 mm P/N 186002161 Insulin HMWP HPLC, 10 μm, 7.8 x 300 mm P/N WAT201549 Empower 3 Auto Blend Plus TM USP EP HMW ACQUITY UPLC BEH125 SEC, 1.7 μm, 4.6 x 300 mm P/N 186006506 0.65 g/l L- 15% 20 % v/v HMW HMW ACQUITY UPLC H-Class Bio Empower 3 USP EP USP P/V HP HV EP USP 0.4% HMW 2.0 EP HMW 120 125 12-12.5 nmusp ACQUITY UPLC BEH125 SEC, 1.7 μm 125 EP SEC 1 BEH125 SEC, 1.7 μm 200 ACQUITY UPLC BEH200 SEC, 1.7 μm HMW 125 200 8% 2 HMW % 0.4% 2 200 P/V=11 125 P/V=37 P/V USP EP 0.4% HMW P/V=2.0 125 HMW HMW Ultra Performance Liquid Chromatography 2
200 125 ACQUITY UPLC BEH125, 1.7 µm Rs=3.37 HMW ACQUITY UPLC BEH200, 1.7 µm Rs=3.13 Rs=2.38 Rs=2.55 4 5 6 7 min 7 8 9 min 1 125 ACQUITY UPLC BEH125 200 ACQUITY UPLC BEH200 HMW SEC 1 2 EP SEC HMW SEC 5-10 μm USP 3-10 μm 1.7 4 10 μm 3 SEC 3 125 300 mm 2 1.7 μm 4 μm 10 μm 35% 2 HMW P/V 3 EP USP 1.7 μm P/V=37 4 μm P/V=9 10 μm P/V=81.7 μm ~5 ml 4 μm ~6 ml 10 μm ~13 ml 3 1.7 μm 10 μm 2 μm UPLC Ultra Performance Liquid Chromatography 3
ACQUITY UPLC BEH125, 1.7 µm Rs=3.37 HMW BioSuite125 UHR, 4 µm Rs=2.21 HMWP, 10 µm (7.8 x 300 mm) Rs=2.08 Rs=1.93 Rs=1.95 Rs=1.92 Rs=1.88 4 5 6 7 min 7 8 9 10 min 14 16 18 20 22 min 21.7 4 10 μm 125HMW SEC 10 μmep 100 μl 0.5 ml/min 1 2 AU 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0.40 0.20 0.00 0 2 4 6 8 10 min 0.83% HMW ACQUITY UPLC BEH125 Insulin 0.000 0.010 0.008 3.8 4.0 0.40 0.20 0.00 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 min 10 20 min Waters HMWP (7.8 x 300 mm) AU 0.006 0.85% HMW 0.004 0.002 0.000 12 13 14 15 16 17 18 19 20 21 min 3 2 ACQUITY UPLC BEH125 Waters HMWP Waters HMWP EP 100 μl 0.5 ml/min ACQUITY UPLC BEH Ultra Performance Liquid Chromatography 4
4.6 x 150 mm 4.6 x 300 mm ACQUITY UPLC BEH125 4 150 mm 300 mm 41-43% 41% 2 2 150 mm 300mm EP USP HMW 2 300 mm 37 150 mm P/V=5 300 mm 150 mm 300 mm 2 ACQUITY UPLC BEH125, 1.7 µm Rs=3.37 ACQUITY UPLC BEH125, 1.7 µm (4.6 x 150 mm) Rs=2.35 HMW Rs=1.87 Rs=1.86 4 5 6 7 min 2.0 2.5 3.0 3.5 min 4 ACQUITY UPLC BEH125 300 mm 150 mm 0.5 ml/min 300 mm 10 μl 150 mm 5 μl Ultra Performance Liquid Chromatography 5
40 35 30 25 Average Peak/Valley Average Rs 3.0 2.5 2.0 P/V 20 1.5 Rs 15 10 1.0 USP/EP Limit (NLT 2) 5 0 ACQUITY UPLC BEH125, 1.7 µm, 300 mm ACQUITY UPLC BEH125, 1.7 µm, 150 mm HMWP, 10 µm, 300 mm Columns BioSuite BEH125, 4 µm, 300 mm ACQUITY UPLC BEH200, 1.7 µm, 300 mm 0.5 0.0 5 HMWP/V USPRs EPUSP P/V HMW USP EP SEC 5 SEC 125 2 μm BEH Waters Ultra Performance Liquid Chromatography UPLC SE- HPLC 1. Walsh G. Biopharmaceutical benchmarks 2010. Nat Biotechnol. 2010; 28:917-24. 2. Insulin, Human, European Directorate for the Quality of Medicines (EDQM), 2001. 3. Insulin, The United States Pharmacopoeia Convention, Inc., Rockville, MD, 2002. www.waters.co.jp 140-0001 1-3-12 5 TEL 03-3471-7191 FAX 03-3471-7118 532-0011 5-14-1011F TEL 06-6304-8888 FAX 06-6300-1734 Waters UPLC ACQUITY UPLC Waters Corporation Empower BioSuite Auto Blend Plus The Science of What s Possible Waters Corporation 2012 Waters Corporation. Printed in Japan. 2012 10 720004271JA PDF