2D-UPLC/MS Catalin Doneanu, Keith Fadgen, StJohn Skilton, Martha Stapels, and Weibin Chen Waters Corporation, Milford, MA, U.S. 4 5 HCP UPLC/MS E HCP
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1 2D-UPLC/MS Catalin Doneanu, Keith Fadgen, StJohn Skilton, Martha Stapels, and Weibin Chen Waters Corporation, Milford, MA, U.S. 4 5 HCP UPLC/MS E HCP DNA ppm HCPHCP HCP HCP 2008 EMA HCP HCP 4 5 ELISA 2 HCP 6 10 HCP 3,4 nanoacquity UPLC with 2D Technology SYNAPT HDMS XBridge and ACQUITY UPLC Columns ACQUITY UPLC Xevo TQ MS HCP2D ph ph MS E MRM 2 3 HCP HCP ELISA HCP 5,6 HCP HCP UPLC /MS LC 1
2 LC 2D-UPLC LC ph D nanoacquity UPLC XBridge TM BEH300C 18 5 µm 1 x 50 mmp/n MS HCP MS SYNAPT HDMS TM 0.5 m/z ESI 2.6 kv 10 µl/ 37 V A 20 mm ph B 5eV ph B 10.8% 12.4% 14.0% % 16.7 % % 20.4% 25.0% 30.0% 50.0% 0.1% TFA / Milli-Q 100 µl/ 15 ev 35 ev MRM LC ACQUITY UPLC ACQUITY UPLC BEH300 C x 150 mm 1.7 µm P/N MS Xevo TQ MS Symmetry C x 20 mm ESI 3.5 kv 5 µm 2.7 µl 37 V LC ph A B ACQUITY UPLC BEH C x 150 mm 1.7 µm P/N µl/ 0.1% / Milli-Q ph % / 7-35% B/30 MS1/MS Da FWHM ProteinLynx Global SERVER TM (PLGS) 2.4 2D-UPLC/MS 2
3 SCX ph 7 2D 2D HCP phrp ph RP HCP MS E / HCP MRM MIX-4 MIX-4 20 mm ph10massprep TM ADH P/N PHO P/N BSA P/N ENL P/N nm ADH 4nM PHO 1 nm BSA 0.2 nm ENL 250 µl PTG1 5 LA PHO ADH BSA ENL 5 10 mg/ml % RapiGest TM mm DTT 30 RT 20 mm IAM 1:20 w/w : Promega 37 TFA 5 µl RapiGest ,000 rpm 10 RapiGest 2 M ph 11 ph 1020 mm ph101 ml 100 µl LA 4000 fmol PHO 800 fmol ADH 320 fmol BSA 80 fmol ENL 16 fmol MRM MRM PTG1 20 nm 250 µl 13 C 15 N 3 Sigma Aldrich HCP 2 CHO IgG1 PTG1 mab 2 A 6 A1 B1 A2 B2 4 DG-44 CHO C D 2 CHO-S 2 A1/A2 B1/B2 2D-UPLC/MS 3
4 2D-UPLC 1A 1C 2D-UPLC ph 101A 10 RP 1B 1:10 0.1% TFA ph C 1A 1B 1C 1. 2D A B ph C ph 2D-UPLC/MS 4
5 20 mmph 10ADH 20 nm PHO 4 nm BSA 1 nm ENL 0.2 nm MIX ph ENLVNQIGTLSESEIK, M+2H 2+ T % 12.4% 15.4% 18.6% 50% B 100% ACN5 T43 ENL % RSD 2D-UPLC 2. 42D-UPLC 18.6% ACN5 4 ENL VNQIGTLSESEIK, [M+2H] 2+ m/z: T % ACN, 0.1% FA12 µl/ ENL 20 fmol 22D-UPLC 31D B % T % RSD 10 ph/ ph 2D 1.9%B 15/10 4 T43 2D-UPLC/MS 5
6 3. RT 4 ENL T43 A 1 1D % ACNB % ACNC % ACND % ACN % ACN, 0.1% FA 4/10 5/10 6/10 4. ph 10 3 ENL T % ACN % ACN % ACN % ACN, 0.1% FA ENL 20 fmole 2D-UPLC/MS 6
7 2D-UPLC 4 ESI-MS / 2D-UPLC HCP 6 10 ph XBridge mab HCP HCP 2D-UPLC/MS E DG-44 A1 A2 B1 B2 CHO-S C D mab PTG1 6 2 A1 mab HCP LC/MS CHO 40 mab 1 PTG1 5 3 HCP 8 1 PTG HCP CHO HCP / HCP ppm 5 4 ENL 3 2 HCP CHO 1HCP / PTG1CHO-S C D HCP DG-44 A1 A2 B1 B2 HCP PTG1A 1 HCP / 2D-UPLC/MS 7
8 ppm DG-44 CHO-S A1 A2 B1 B2 C D 1 Nucleolin Mesocricetus auratus Mus musculus Mus musculus C 1 Mus musculus Mesocricetus auratus Mus musculus b PHO RNA Mus musculus Mesocricetus auratus kda Mesocricetus auratus Mus musculus Mus musculus T 1 Mus musculus HTRA1 Mus musculus Mus musculus S S3 Mus musculus LA Mus musculus Mus musculus M2 Mus musculus RNA II p HSP 90 Mus musculus Mus musculus Mus musculus C1q Mus musculus ADH Mus musculus Mus musculus Mus Musculus BSA Mus musculus Mus musculus kda Mus musculus HSP 90 Mus musculus Mus musculus HCP ppm PTG ESI-MS 3PTG1 mab 6HCPppm 1 g HCP A1 A2CProSep-vAB1 B2DMabSelect Sure A1/A2B1/B2 DG-44 CDCHO-S mab 2D-UPLC/MS 8
9 UPLC/ MS HCP HCP UPLC/MRM-MS ELISA UPLC/MRM/MS 2D-UPLC/MS E 20 UPLC/MRM-MS UPLC/MRM-MS HCP 6 HCP 20 MRM HCP MRM RSD 13.8% UPLC/MRM-MS HCP MRM HCP UPLC/MS MRM HCP 20 HCP 3 MRM 5A C 6 MS E Conc (ppm) A1 A2 B2 (A) MS E MRM Conc (ppm) B1 B2 (B) MS E MRM Conc (ppm) A2 (C) B1 C A A1 B1 B2 MS E MRM D 300 D 400 A C 200 C 100 D MS E MRM HCP TOF MS E 3 MRM 13 C 15 N mab 6 ppm A B1C 3 2D-UPLC/MS 9
10 UPLC/MS E 4 5 HCP mab HCP mab A HCP HCP UPLC/MRM-MS 1. European Medicines Agency. Omnitrope: Scientific Discussion pdf 2. Wang X, Hunter AK, Mozier NM. Biotech & Bioeng. 2009: 103, Hayduk EJ, Choe LH, Lee KH. Electrophoresis. 2004: 25, Jin M, Szapiel N, Zhang J, Hickey J, Ghose S. Biotech & Bioeng. 2010: 105, Savino E, Hu B, Sellers J, Sobjak A, Majewski N, Fenton S, Yang TY. BioProcess Intl. 2011: 9, Savino E, Hu B, Sellers J, Sobjak A, Majewski N, Fenton S, Yang TY. BioProcess Intl. 2011: 9, Wolters DA, Washburn MP, Yates JR III. Nat Biotechnol. 2001: 19, Li GZ, Vissers JP, Silva JC, Golick D, Gorenstein MV, Geromanos SJ. Proteomics. 2009: TEL FAX F TEL FAX Waters ACQUITY UPLC UPLC nanoacquity UPLC SYNAPT Xevo Waters Corporation XBridge MassPREP RapiGest ProteinLynx Global SERVER The Science of What s Possible Waters Corporation 2012 Waters Corporation. Produced in Japan JA PDF
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