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1 HPLC :4: 8

2 5, ProODSYMC-Pack ODS 5,~, Wide-Pore ColumnsYMCbasicPROTEIN-RP YMC-BioPro YMC-Pack Diol YMC-Pack Diol-NP Hydrosphere C8YMC-Pack ODS-AQ Hydrosphere C8YMC-Pack ODS-AQ Wide-Pore Columns YMC-BioPro YMC-Pack Diol YMC-Pack Diol-NP YMC-Pack Polyamine

3 BSA N88B min N84B min N86L min Column : YMCbasic 5 m, 5 X. mmi.d. Eluent : A) water / TFA (/.) B) acetonitrile / TFA (/.) 5-5%B (-5 min), 5-45%B (5-55 min), 45%B (55-6 min) Flow rate :. ml/min Temperature : 7 Detection : UV at nm Injection : L Column : YMC-BioPro QA 5 m, 5 X 4.6 mmi.d. Eluent : A) mm Tris-HCl (ph 8.6) B) mm Tris-HCl (ph 8.6) containing.5 M NaCl -5%B (- min), 5-6%B (-6 min) Flow rate :.5 ml/min Temperature : 5 Detection : UV at nm Injection : L Column : YMC-Pack Diol- Diol-6 5 m, 5 X 8. mmi.d. X Eluent :. M KHPO4-KHPO4 (ph 7.) containing. M NaCl / acetonitrile (7/) Flow rate :.7 ml/min Temperature : ambient (5) Detection : UV at nm Injection : 5 L

4 4

5 Reversed Phase Chromatography (RPC) ~ m C8, C8, C4 etc..% TFA / CH CN etc CHCN () Time 5

6 Angiotensin II (MW,46) \ \ nm nm BSA (MW 66,) \ \ nm nm 6

7 6 nm nm Insulin Chain B, Oxidized (MW,495) BSA (MW 66,) Efficiency (plates/m) 4 C4 C8 C8 C4 C8 C8 C8 C8 C4 C4 C8 C Mass (mg) Mass (mg) 7

8 C8 C8 C4 nm 5, nm,, nm : excellent : good : moderate 8

9 YMC (nm) Pro Pro C8 ODS Hydrosphere C8 ODS Pro C8 RS ODS 8 C8 C4 YMC-Pack ODS-A ODS ODS-AQ ODS C8 C4 Ph CN YMCbasic PROTEIN-RP Polymer C8 9

10 m YMC-UltraHT -Lactoglobulin B YMC-Pack Pro C8, 5 m X. mmi.d PC = MPa (N75D) 5 m 5 min YMC-UltraHT Pro C8, m X. mmi.d PC = MPa (N75G) 5 PC (peak capacity) = (gradient time / peak width*) *peak width = W.5h average Eluent : A) water / TFA (/.) B) acetonitrile / TFA (/.) 5-5%B (-5 min) Flow rate :.4 ml/min Temperature : 7 Detection Injection Sample : UV at nm : L : Tryptic digest of -Lactoglobulin B (5 mg/ml; 7, 4 h) 5 min undigested -Lactoglobulin B C8, nm m

11 (MW 4,7,) () Model casepeptides & small proteins Amyloid -protein (-4) MW 4, Insulin, from Bovine pancreas MW 5,7 Cytochrome c, from Horse heart MW,4 -Lactalbumin, from Human milk MW 4, Lysozyme, from Egg white MW 4, Myoglobin, from Horse skeletal muscle MW 7,.. or. C8, nm 5 m 5 X 4.6 mmi.d. Column : YMC-Pack C8 (5 m, nm) 5 X 4.6 mmi.d. Eluent : A) water / TFA (/.) B) acetonitrile / TFA (/.) Flow rate :. ml/min Temperature : 7 Detection : UV at nm

12 (MW 4,7,) () %B (- min) %B (- min) %B (- min) %B (- min)

13 (MW 4,7,) () 5 7%B (- min) %B (- min) %B (- min) %B (- min) 4 5 6

14 . Cytochrome c MW,4. Insulin MW 5,7. Amyloid -protein MW 4, 4. Lysozyme MW 4, 5. -Lactalbumin MW 4, 6. Myoglobin MW 7, (MW 4,7,) (4) C8, nm Column : 5 m, 5 X 4.6 mmi.d. Eluent : A) water / TFA (/.) B) acetonitrile / TFA (/.) 5-6%B (- min) Flow rate :. ml/min Temperature : 7 Detection : UV at nm C8, nm C8, nm 4 5 6, C8, nm C4, nm

15 (MW 66,96,) () Model caseproteins (MW 66,-96,). BSA MW 66,. Conalbumin MW 77,. Lipoxidase MW 96, Column Flow rate Temperature Detection : C4 (5 m, nm) 5 X 4.6 mmi.d. :. ml/min : 7 : UV at nm 5

16 (MW 66,96,) () -Propanol A) water / TFA (/.) B) acetonitrile / TFA (/.) A) water / TFA (/.) B) acetonitrile / -propanol / TFA (5/5/.) (TPSAMP D) (TPSAMP D) 5 5 min 5 5 min -75%B (-5 min), 75%B (5- min) 6

17 (MW 66,96,) (). BSA MW 66,. Conalbumin MW 77,. Lipoxidase MW 96, C4, nm Column : 5 m, 5 X 4.6 mmi.d. Eluent : A) water / TFA (/.) B) acetonitrile / -propanol / TFA (5/5/.) -75%B (-5 min), 75%B (5- min) Flow rate :. ml/min Temperature : 7 Detection : UV at nm (TPSAMP D) min C4, nm C8, nm (TPSAMP.4 57.D) (TPSAMP D) C4, nm C8, nm (TPSAMP D) (TPSAMP.4 58.D) min min 7

18 H C NH CH O HN O CH CH Ō P O O O O N H C O H C H N Ō O P O HN CH H C O O O N O H C O HN CH Ō P O NH CH CH O O O N H C NH CH O CH 8

19 Hydrosphere C8 Hydrosphere C8, mm TEAA T T T J59A YMC-Pack Pro C8, mm TEAA T J5E Oligodeoxythymidylic acid [d(t)-] Column : 5 m, 5 X 4.6 mmi.d. Eluent : A) mm or mm TEAA* (ph 6.) B) mm or mm TEAA* (ph 6.) / acetonitrile (8/) 55-6%B (- min) Flow rate :. ml/min Temperature : 5 Detection : UV at 69 nm Injection : 5 L (5 nmol/ml) triethylamine-acetic acid T- Brand I, mm TEAA T J59B Brand I, mm TEAA T Hydrosphere C8 J54A 9

20

21 Ion Exchange Chromatography (IEC) 5 mmnacl NaCl Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl NaCl (M) Time

22 - pi ph pi ph pi.5.phph 57 pi.5.phph 79 piph DNA

23 YMC-BioPro QASP YMC-BioPro QA YMC-BioPro SP YMC-BioPro QA-F YMC-BioPro SP-F (m) 5 (nm) Non-porous -CH N (CH ) -CH CH CH SO - -CH N (CH ) -CH CH CH SO - (meq/ml-resin) (mg/ml-resin) 5 (BSA) 7 (human IgG) (BSA) 8 (human IgG) () ph 46

24 YMC-BioPro () DBC (Dynamic binding capacity) : DBC DBC for BSA Eluted amount Recovery* (mg/ml-resin, % breakthrough) (mg/ml-resin) (%) YMC-BioPro QA 6 95 A (porous Q type) B (porous Q type) 5 5 *Recovery : (Eluted amount / DBC) X Column : YMC-BioPro QA 5 X 4.6 mmi.d. A (porous Q type) 5 X 4.6 mmi.d. B (porous Q type) 5 X 5. mmi.d. Linear velocity :. cm/min Sample : mg/ml BSA in equilibration buffer Equilibration buffer : mm Tris-HCl (ph 8.6) Elution buffer : mm Tris-HCl (ph 8.6) containing. M NaCl Detection : UV at 8 nm YMC-BioPro 4

25 YMC-BioPro () DBC DBC (mg/ml-resin) 5 5 YMC-BioPro QA Trypsin inhibitor BSA IgG DBC (mg/ml-resin) 5 5 YMC-BioPro SP Lysozyme Avidin IgG.5..5 (X 5 ).5..5 (X 5 ) Protein MW DBC Eluted amount Recovery Protein MW DBC Eluted amount Recovery (mg/ml-resin) (mg/ml-resin) (%) (mg/ml-resin) (mg/ml-resin) (%) IgG 5, 4 9 IgG 5, BSA 66, 6 95 Avidin 68, 9 9 Trypsin inhibitor, 7 95 Lysozyme

26 YMC-BioPro () YMC-BioPro QA 5 m, 5 X 4.6 mmi.d. N86B N87C N87B N86A min 5 g 5 g g 5 g. Ovalbumin. Trypsin inhibitor Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing.5 M NaCl -8%B (- min) Flow rate :.5 ml/min Temperature : 5 Detection : UV at 8 nm Injection : L 5 X 7 4 X 7 X 7 B (porous Q type) m, 5 X 5. mmi.d. X 7 X N86E N88C N88B N86D 5 g 5 g g min 5 g (mg)trypsin inhibitor YMC-BioPro QA B (porous Q type) YMC-BioPro QA 6

27 YMC-BioPro (4) YMC-BioPro QA 5 m, 5 X 4.6 mmi.d. Albumin IgG Transferrin min pi Albumin (MW 66,) Transferrin (MW 75,) 5. IgG (MW 5,-) A (porous Q type) m, 5 X 4.6 mmi.d. min B (porous Q type) m, 5 X 5. mmi.d. min Eluent : A) mm Tris-HCl (ph 8.6) B) mm Tris-HCl (ph 8.6) containing.5 M NaCl -%B (-5 min), -%B (5- min) Flow rate :.5 ml/min Temperature : 5 Detection : UV at 8 nm Injection : L Sample : Human serum ( L/mL) YMC-BioPro DBC 7

28 YMC-BioPro () mm YMC-BioPro SP-F 5 m, X 4.6 mmi.d.. Ribonuclease A. Cytochrome c. Lysozyme AU.4..5 ml/min MPa Eluent : A) mm KHPO4-KHPO4 (ph 6.8) B) mm KHPO4-KHPO4 (ph 6.8) containing.5 M NaCl -%B (-4 min) for YMC-BioPro SP-F -%B (-6 min) for YMC-BioPro SP Temperature : 5 Detection : UV at nm Injection : L N7K 4 min YMC-BioPro SP 5 m, 5 X 4.6 mmi.d. AU YMC-BioPro.5.5 ml/min.-. MPa N76E 4 6 min 8

29 YMC-BioPro () YMC-BioPro SP-F 5 m, X 4.6 mmi.d. AU.4. N7K A (non-porous S type).5 m, 5 X 4.6 mmi.d. AU.4. N7J N() 7 N() N() 76 Rs(, ) 7.6 Rs(, ) MPa 4 min N() 44 N() 6 N() 4 Rs(, ) 4.5 Rs(, ) MPa 4 min. Ribonuclease A. Cytochrome c. Lysozyme Eluent : A) mm KHPO4-KHPO4 (ph 6.8) B) mm KHPO4-KHPO4 (ph 6.8) containing.5 M NaCl -%B (-4 min) for YMC-BioPro SP-F -%B (-4.67 min) for A (non-porous S type) Flow rate :.5 ml/min Temperature : 5 Detection : UV at nm Injection : L (. mg/ml) YMC-BioPro 9

30 YMC-BioPro () 4 NaN MAb YMC-BioPro QA-F 5 m, X 4.6 mmi.d..5 MPa MAb : Mouse monoclonal IgG (Anti-human IgG4) P8A min 4 NaN MAb A (non-porous DEAE type).5 m, 5 X 4.6 mmi.d. 7.5 MPa P89A min Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing.5 M NaCl -5%B (-8 min) Flow rate :. ml/min Temperature : 5 Detection : UV at nm Injection : L (. mg/ml) Sample : Monoclonal mouse IgG (Purified by DEAE chromatography, containing NaN) 4 NaN B (non-porous Q type) m, 5 X 4.6 mmi.d. MAb YMC BioPro QA-F 5.9 MPa P8B min

31 YMC-BioPro (4) mm mm YMC-BioPro QA-F mm 4 Lot. A MAb 4 Lot. A MAb P8A min P844A 4 min Lot. B Lot. B MAb 4 MAb 4 P847A P844B min 4 min Column : YMC-BioPro QA-F X 4.6 mmi.d. YMC-BioPro QA-F X 4.6 mmi.d. Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing.5 M NaCl -5%B (-8 min) for mm length column -5%B (-6 min) for mm length column Flow rate :. ml/min Temperature : 5 Detection : UV at nm Injection : L for mm length column 4 L for mm length column Sample : Monoclonal mouse IgG (. mg/ml) (Purified by DEAE chromatography, containing NaN) MAb : Mouse monoclonal IgG (Anti-human IgG4) mm

32 YMC-BioPro QA-F 5 m, X 4.6 mmi.d. 5 X X / DNA NaCl N8D 5 min 5 Step ).5-. M NaCl (-5 min) Slope. M NaCl/min Step ).5-. M NaCl (-5 min) Slope. M NaCl/min Eluent : A) mm Tris-HCl (ph 8.) containing.5 M NaCl B) mm Tris-HCl (ph 8.) containing. M NaCl Flow rate :.5 ml/min Temperature : 5 Detection : UV at 6 nm Injection : L (.5 mg/ml) Sample : kb DNA ladder (75-,6 bp) N8E 4 5 min 5 NaCl. M Step ).7-. M NaCl (- min) Slope. M NaCl/min N8G min

33 mm YMC-BioPro QA-F YMC-BioPro QA-F X 4.6 mmi.d. 5 Eluent : A) mm Tris-HCl (ph 8.) containing.7 M NaCl B) mm Tris-HCl (ph 8.) containing. M NaCl -%B (- min) Flow rate :.5 ml/min Temperature : 5 Detection : UV at 6 nm Injection : L (.5 mg/ml) Sample : kb DNA ladder (75-,6 bp) N8G 5 5 min YMC-BioPro QA-F X 4.6 mmi.d. 5 YMC-BioPro QA-F mm N8B 5 5 min

34 4

35 Size Exclusion Chromatography (SEC) 5~ m phbuffer, ph 7 Time 5

36 MW (Calibration curve) 隙 Log MW Elution volume SEC 6

37 YMC-Pack Diol 4 Diol-6 Diol- Diol- Diol- (m) (Diol 5 (nm) 6 ph () , 7

38 MW (MW 9 MW Diol- 6 5 Diol Diol MW. IgM 9,. Thyroglobulin 67,. IgA 9, 4. Fibrinogen 4, 5. -Globulin 58, 6. IgG 5, 7. Transferrin 75, 8. HSA* 66, 9. -Antitrypsin 5,. Ovalbumin 45,. Carbonic anhydrase,. Trypsin inhibitor,. Myoglobin 7, 4. -Lactalbumin 4, 5. Ribonuclease A,7 6. Cytochrome c, Elution volume (ml) Column : YMC-Pack Diol X 8. mmi.d. Eluent :. M KH PO 4 -K HPO 4 (ph 7.) containing. M NaCl Flow rate :.5 ml/min Temperature : 5 Detection : UV at 8 nm *HSA : Human serum albumin Diol- MW Diol- MW Diol- MW 5, 8

39 MW MW Diol Elution volume (ml) Column : YMC-Pack Diol-6 5 X 8. mmi.d. Eluent :. M KH PO 4 -K HPO 4 (ph 7.) containing. M NaCl / acetonitrile (7/) Flow rate :.7 ml/min Temperature : ambient Detection : UV at 5 nm 7 MW. Myoglobin 7,. Ribonuclease A,7. Cytochrome c,4 4. Insulin (bovine) 5,7 5. Insulin chain B, Mating factor, Neurotensin,67 8. Angiotensin I,96 9. CCK-Octapeptide,4. Bradykinin,6. Angiotensin II,4. Oxytocin,7. Angiotensin III 9 4. Met-Enkephalin Leu-Enkephalin Tetraglycine Glycine 75 Diol-6 MW 9

40 YMC-Pack Diol- 5 m, X 8. mmi.d. () YMC-Pack Diol- 5 m, X 8. mmi.d. IgG (MW 5,) Transferrin (MW 75,) Albumin (MW 66,) N869A min N869O min YMC-Pack Diol- Diol- 5 m, X 8. mmi.d. X Diol-Diol- Eluent :. M KH PO 4 -K HPO 4 (ph 7.) containing. M NaCl Flow rate :.5 ml/min Temperature : ambient (5) Detection : UV at 8 nm Injection : L Sample : Human serum ( L/mL) N866A min IgGIgGTransferrinAlbumin 4

41 () BSA YMC-Pack Diol-6 5 m, 5 X 8. mmi.d. 5 N86L min Eluent :. M KH PO 4 -K HPO 4 (ph 7.) containing. M NaCl / acetonitrile (7/) Flow rate :.7 ml/min Temperature : ambient (5) Detection : UV at nm Injection : 5 L Sample : Tryptic digest of BSA Diol-Diol-6 YMC-Pack Diol- Diol-6 5 m, 5 X 8. mmi.d. X N864F min 4

42 (Injection volume) () IgG. mg Injection L L Sample. mg/ml. mg/ml N() N() 78 8 Rs(, ).6.8 P885A P886A 4 Injection : L Sample :. mg/ml Injection : L Sample :. mg/ml min. mg Injection L L Sample mg/ml. mg/ml N() N() Rs(, ).57. P887A 4 P887B Column : YMC-Pack Diol- Diol- X 4.6 mmi.d. X Eluent :. M KHPO4-KHPO4 (ph 7.) containing. M NaCl Flow rate :.7 ml/min Temperature : ambient (5) Detection : UV at 8 nm. IgG (MW 5,). Albumin (MW 66,) Injection : L Sample : mg/ml Injection : L Sample :. mg/ml SEC min 4

43 (Injection volume) () Injection L ( L/mL) N() N() 7 Injection L ( L/mL) N() 4 N() 4 YMC-Pack Diol- Diol- 5 m, X 4.6 mmi.d. X, Eluent Flow rate. IgG (MW 5,). Transferrin (MW 75,). Albumin (MW 66,) :. M KHPO4-KHPO4 (ph 7.) containing. M NaCl :.7 ml/min P888A P888B min Temperature : ambient (5) Detection : UV at 8 nm YMC-BioPro QA 5 m, 5 X 4.6 mmi.d. 8 Injection L ( L/mL) N() 9 N() 8 Eluent : A) mm Tris-HCl (ph 8.6) 6 4 Injection L ( L/mL) N() 5 N() 5 P889A P889B min B) mm Tris-HCl (ph 8.6) containing.5 M NaCl -%B (-5 min), -%B (5- min) Flow rate :.5 ml/min Temperature : 5 Detection : UV at 8 nm 4

44 Pro, Wide-Pore etc. YMC-BioPro YMC-Pack Diol or, ph, ph 44

45 (IgG) 45

46 5 5 YMC-Pack Diol- Diol- 5 m, X 8. mmi.d. X Albumin Transferrin IgG Eluent :. M KHPO4-KHPO4 (ph 7.) containing. M NaCl Flow rate :.5 ml/min Temperature : ambient (5) Detection : UV at 8 nm Injection : L ( L/mL) N866A min YMC-BioPro QA 5 m, 5 X 4.6 mmi.d. IgG Albumin Transferrin Eluent : A) mm Tris-HCl (ph 8.6) B) mm Tris-HCl (ph 8.6) containing.5 M NaCl -%B (-5 min), -%B (5- min) Flow rate :.5 ml/min Temperature : 5 Detection : UV at 8 nm Injection : L ( L/mL) N8E min TransferrinAlbumin TransferrinAlbumin 46

47 (MAb) YMC-Pack Diol- 5 m, X 4.6 mmi.d. 4 MAb NaN Eluent :. M KHPO4-KHPO4 (ph 7.) Flow rate :.7 ml/min Temperature : ambient (5) Detection : UV at nm Injection : L Sample : a commercially available mouse monoclonal IgG (.5 mg/ml) (purified by DEAE chromatography, containing NaN ) P85A min YMC-BioPro QA-F 5 m, X 4.6 mmi.d. 5 4 NaN MAb P8A min Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing.5 M NaCl -5%B (-8 min) Flow rate :. ml/min Temperature : 5 Detection : UV at nm Injection : L Sample : a commercially available mouse monoclonal IgG (. mg/ml) (purified by DEAE chromatography, containing NaN) MAb = Mouse monoclonal IgG (Anti-human IgG4) DEAEMAb (IgG) 47

48 IgG YMC-Pack Diol- 5 m, X 8. mmi.d. IgG Fc fragment 6 4 IgG Eluent :. M KHPO4-KHPO4 (ph 6.9) containing. M NaCl Flow rate :. ml/min Temperature : ambient (7) Detection : UV at nm Injection : 5 L (.5 mg/ml) R86D 5 5 min YMC-Pack C4 ( nm) 5 m, 5 X 4.6 mmi.d. IgG Fc fragment Eluent : A) water / TFA (/.) B) acetonitrile / TFA (/.) 5-45%B (-4 min) Flow rate :. ml/min Temperature : 7 Detection : UV at nm Injection : 5 L (. mg/ml) R869B min IgG IgG 48

49 NH O O 4 O 5 NH N CH N HN HN N HN N O NH O NH H N NH N O NH N NH Cytosine Uracil Guanine Thymine Adenine 4 5 Column : Hydrosphere C8 5 m, 5 X 4.6 mmi.d. Eluent : mm KHPO4-KHPO4 (ph 6.9) Flow rate :. ml/min Temperature : 7 Detection : UV at 54 nm,.64 AUFS Injection : 8 L (.-. mg/ml) min S999A UTP. 5 -UDP. 5 -CMP UMP dcmp GMP ATP IMP ADP. 5 -TMP. 5 -AMP Column : Hydrosphere C8 5 m, 5 X 4.6 mmi.d. Eluent : mm KHPO4-KHPO4 (ph 5.5) Flow rate :. ml/min Temperature : Detection : UV at 6 nm,.56 AUFS Injection : L (.5-.5 mg/ml) % Hydrosphere C8 min A9A 49

50 d(t) - LC/MS UV % Oligodeoxythymidylic acid [d(t)-] T T Diode Array 69 nm 5.5e4 T MS % T T T Scan ES- TIC 7.5e7 5 5 min Column : Hydrosphere C8 m, 5 X. mmi.d. Eluent : A) 5 mm di-n-butylamine-acetic acid (ph 6.) B) 5 mm di-n-butylamine-acetic acid (ph 6.) / methanol (/8) 4-7%B (- min) Flow rate :. ml/min Temperature : 5 Detection : UV at 69 nm ESI negative-mode Injection : 5 L (5 nmol/ml) LC/MS Hydrosphere C8 bufferlc/ms 5

51 pbrhae 4 YMC-Pack Diol- Diol- 5 m, 5 X 8. mmi.d. X. Plasmid pbr Hae digest (8-587 bp). Plasmid pbr (4,6 bp) P867A 4 5 min Eluent :. M KHPO4-KHPO4 (ph 7.) containing. M NaCl Flow rate :.7 ml/min Temperature : ambient (5) Detection : UV at 6 nm Injection : L Sample : Plasmid pbr Hae digest (. mg/ml) Plasmid pbr (. mg/ml) YMC-BioPro QA-F 5 m, X 4.6 mmi.d. 4 Eluent : A) mm Tris-HCl (ph 8.) B) mm Tris-HCl (ph 8.) containing. M NaCl 7-85%B (- min), 85%B (-5 min) Flow rate :.5 ml/min Temperature : 5 Detection : UV at 6 nm Injection : L Sample : Plasmid pbr Hae digest (. mg/ml) Plasmid pbr (. mg/ml) N86F min 5

52 ph 5

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