SEC (SEC) SEC SEC SEC 2
SEC ( 1) 1: SEC www.agilent.com/chem/jp 3
( ) ( 2) ICH (Q6B) 3 lgg 8 7 6 5 4 1. 2. 3. 4. 5. Agilent AdvanceBio SEC 3 Å 7.8 x 3 mm 2.7 µm (p/n PL118-531) Agilent 126 Infinity LC 1. ml/min 3 2 2 1 1 5 4 2 4 6 8 1 12 14 2: IgG min UV 22 nm 5 µl IgG 15 mm ph 7. SEC ( 3) ( ) 3: 4
SEC-UV/DAD ( ) mab MW >.1 1,25 kda mab MW >.1 1, kda AdvanceBio SEC (2.7 μm) MW (kda) 13 Å.1 1 3 Å 5 1,25 Agilent Bio SEC-5 (5 m) MW (kda) 1 Å.1 1 15 Å.5 15 3 Å 5 1,25 5 Å 15 5, 1 Å 5 7,5 2 Å > 1, AdvanceBio SEC Agilent Bio SEC-5 15 mm ph 7.* 1 3 1 3 ºC 8 C 4.6 mm.1.4 ml/min 7.8 mm.1 1.25 ml/min 5 % * Defining the Optimum Parameters for Efficient Size Separations of Proteins ( ) ( 599-8895EN) www.agilent.com/chem/jp ph.2 5 mm ph 7. 1 15 mm 5 mm ph 7. 1 15 mm 5 mm ph 7. 5 1 mm (KCl ) ph 2. 8.5 5 mm ph 7. 5 1 % ( ) 5 mm ph 7. 5 % DMSO SEC 1 3 C SEC Agilent Bio SEC 8 C 5
SEC (RI) UV DAD RI 2 M NaCl 8 M ph 1 13 ( ) LC 126 Infinity LC LC Agilent www.agilent.com/chem/jp Agilent 126 Infinity LC RFID 1 mm 13 µl (p/n G5615-622) 6
HPLC Agilent OpenLAB CDS ChemStation GPC/SEC Agilent GPC/SEC Buffer Advisor ph ph SEC ( ) ( ) ( 4) SEC 17 Mn Mw Mp. 2. 4. 6. 8. 1. 12. 14. 4: SEC Mw Mn Mp 7
HPLC HPLC ( ) ( ) ( ) ( ) 5 Captiva PES LC PVDF PES PES LC PVDF www.agilent.com/chem/jp Captiva PES (mm) (μm) 4.45 LC 519-595 4.2 LC/MS 519-594 15.2 LC/MS 519-596 15.45 LC 519-597 25.2 LC/MS 519-598 25.45 LC 519-599 8
SEC ( ) SEC 7.8 x 3 mm 1. ml/min 2.1 4.6 x 15 mm SEC 2 3 SEC SEC SEC (5 2 µl) (1 4 mg/ ml) 1 1 12 ( 7.8 x 3 mm 1. ml/min ) HPLC SEC 2 3 mm 15 mm 5 7 6 5 4 3 2 1 AdvanceBio SEC 7.8 x 3 mm 1. ml/min IgG 1 2 3 1. 2. 3. 4. 5. 2 4 6 8 1 12 4 14 5 分 AdvanceBio SEC 7.8 x 15 mm 2. ml/min IgG 6 3 5 4 3 3.5 2 1 1 2 5 4.5 1 1.5 2 2.5 3 分 5: 3 mm 15 mm 9
( ) ( 1) / (SEC) SEC-UV/DAD LS mab SEC-MS mab Agilent AdvanceBio SEC Agilent Bio SEC-3 Agilent Bio SEC-5 MS (1 Å 15 Å 3 Å 5 Å 1 Å 2 Å) ProSEC 3S ZORBAX GF-25/45 USP L35 PEG 2 3 PL aquagel-oh ü PL aquagel-oh 8 µm ü PL aquagel-oh 2 5 µm ü PL aquagel-oh MIXED-M 8 µm 2 3 PL aquagel-oh ü PL aquagel-oh MIXED-H 8 µm ü PL aquagel-oh 6/5/4 8 µm PL aquagel-oh 6/5/4 15 µm PL aquagel-oh ph 2 1 ( 5 % ) 14 bar (23 psi) 1: Agilent Bio SEC PL Aquagel-OH 1
3 Å 6 5 IgG 3 Å IgG BioRad 2 175 15 125 1 75 5 25 A: B: A 3 1 2 4 2 4 6 8 1 12 14 分 AdvanceBio SEC 3 Å 4.6 x 3 mm 2.7 µm (p/n PL158-531) AdvanceBio SEC 13 Å 4.6 x 3 mm 2.7 µm (p/n PL158-535) 1. 2. g- ( ) 3. ( ) 4. ( ) 5. B12 5 6 B 1 5 4 3 2 2 3 4 5 1 2 4 6 8 1 12 14 Agilent 126 Infinity LC 15 mm ph 7..35 ml/min UV 22 nm 分 BioRad IgG 3 1. 7 A 2. 6 3. 5 4. 5. 4 3 2 2 1 1 4 5 2 4 6 8 1 12 14 分 A: AdvanceBio SEC 3 Å 4.6 x 3 mm 2.7 µm (p/n PL158-531) B: AdvanceBio SEC 13 Å 4.6 x 3 mm 2.7 µm (p/n PL158-535) 1 2, 3 B 8 6 1 4 2 5 4 2 4 6 8 1 12 14 Agilent 126 Infinity LC 15 mm ph 7..35 ml/min UV 22 nm 分 IgG 6: BioRad IgG 2 11
SEC SEC / / PEG/PEO ( 7) / PEG/PEO / 3 ph 8 5, PEG 7 7.5 9.5 SEC 5, / 17 (g/mol) 4, 3, 2, 1, 1,, 1, 1, 1, PEG/PEO / 1 4 5 6 7 8 9 1 11 12 13 (ml) 7: 3 PEG 46,72 ( MV) 48, ( MV) 44,3 ( MV) 2 4 6 8 1 分 8: 13 Å AdvanceBio SEC (p/n 519-9416 13 Å AdvanceBio SEC 2 ml ) 5 ( II) 13 Å Agilent AdvanceBio 3 Å AdvanceBio SEC (p/n 519-9417 3 Å AdvanceBio SEC 2 ml ) 5 ( g- II) 3 Å Agilent AdvanceBio 12
( / ) 9 3 µm Agilent Bio SEC-3 5 µm Agilent Bio SEC-5 Agilent Bio SEC-3 Agilent Bio SEC-5 Bio SEC-3 3 Å 7.8 x 3 mm 3 µm (p/n 519-2511) Bio SEC-5 3 Å 7.8 x 3 mm 5 µm (p/n 519-2526) Agilent 126 Infinity LC 15 mm ph 7 1 ml/min UV 22 nm 12 1 8 6 4 2 1 1 2 Rs 2 3 3 1. 2. 3. 3 µm 5 µm 2 4 6 8 1 12 14 16 18 分 9: Agilent Bio SEC-3 Agilent Bio SEC-5 3 µm 4.6 mm (.35 ml/min) SEC 21.2 mm Agilent SEC-3 SEC-5 HPLC Agilent AdvanceBio SEC 7.8 x 3 mm 4.6 x 3 mm 13
3 mm 15 mm SEC 1 15 mm 2 ml/min 4 IgG 14 12 1 8 6 4 2 A 2 4 6 8 1 12 14 分 AdvanceBio SEC 3 Å 7.8 x 15 mm 2.7 µm (p/n PL118-331) 15 mm ph 7..5 1. 1.5 ml/min (52 12 152 bar) UV 22 nm 5 µl 正規化後 14 12 1 8 6 B.5 ml/min 1. ml/min 1.5 ml/min IgG (2 mg/ml) 4 2 2 4 6 8 1 12 14 分 1: 12 4 (A) (B) SEC (1 2 %) ( ) ( ) 5 1 mm Agilent 126 Infinity LC / 11 14
(ph) ( 11) 3 25 2 15 1 5 3 A 2 4 6 8 1 12 14 B 5 µl Agilent Bio SEC-3 3 Å 4.6 mm x 3 mm 3 µm (p/n 519-2513) Agilent 126 Infinity LC.35 ml/min UV 22 nm A: 2 mm ph 7 + 5 mm NaCl B: 2 mm ph 7 + 1 mm NaCl C: 2 mm ph 7 + 4 mm NaCl (1 mg/ml 2 mm ph 7) 2 mm 5 mm NaCl 25 2 15 1 5 2 4 6 8 1 12 14 C 3 25 2 15 1 5 2 mm 4 mm NaCl 2 4 6 8 1 12 14 11: 15
( 12) Agilent Bio SEC-5 7.8 x 3 mm 5 µm (p/n 519-2521) Agilent 126 Infinity LC 15 mm ph 7. 1. ml/min UV 1,, 1, MW 1 Å 5 Å 3 Å 15 Å 1 Å 67, 1.7 8.23 7.3 5.82 5.77 g- 158, 1.88 9.8 8.57 6.55 5.79 1, 1 Å 5 Å BSA 67, 11.13 1.44 9.44 7.29 6. 45, 11.28 1.83 9.89 7.9 6.4 1, 3 Å 17, 11.44 11.28 1.42 8.66 7.5 15 Å A 12,7 11.52 11.41 1.58 8.93 7.32 1 4 1 Å 5 6 7 8 9 1 11 12 13 (ml) B12 1,35 12. 12.59 11.78 11.49 1.3 112 12.8 12.68 12.21 12.13 11.41 12: 16
SEC UV (DAD) ( ) 22 nm 254 nm 28 nm UV RI SEC ( 13) Agilent AdvanceBio 3 Å 7.8 x 3 mm 2.7 μm 45 Agilent 126 Infinity LC Agilent 126 Infinity GPC/SEC 4 35 3 RI LS 9 UV, 28 nm 15 mm ph 7..8 ml/min 3 C UV 28 nm + RI + LS 9 レスポンス (mv) 25 2 15 1 5 µl 5-5 -1 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 RT ( 分 ) 13: 17
/ (ADC) SEC ADC AdvanceBio SEC 14 正規化後 175 15 125 1 75 5 25 DAD1 A, Sig=22 インタクトトラスツズマブ DAD1 A Sig=22 熱/pH ストレスを加えたトラスツズマブ 凝集体 8.49 インタクトトラスツズマブフラグメント 6.61 7.33 11.584 12.74 13.114 14.469 2 4 6 8 1 12 14 分 16 14 12 1 8 6 4 2 DAD1 A, Sig=22 インタクト ADC DAD1 A Sig=22 熱/pH ストレスを加えた ADC ADC 凝集体 6.654 7.141 8.16 8.67 インタクト ADC 11.586 ADC 分解物 12.737 2 4 6 8 1 12 14 分 AdvanceBio SEC 3 Å 7.8 x 3 mm 2.7 µm Agilent 126 Infinity LC PBS 15 mm 5 mm ph 7.4 TCC 1 µl.8 ml/min UV 22 nm 14: mab ADC www.agilent.com/chem/navigator LC 4 LC USP 18
SEC 25 mm 3 mm 7.5 7.8 mm 1. ml/min 4.6 mm.35 ml/min 15 mm ( ) / / 7 ( ) ( ) ph ( ) SEC www.agilent.com/chem/jp 19
agilent.com/chem/jp www.agilent.com/chem/jp 12-477-111 email_japan@agilent.com Agilent Technologies, Inc. 215 Printed in Japan November 1, 215 5991-3651JAJP