SEC (SEC) SEC SEC SEC 2

Size: px

Start display at page:

Download "SEC (SEC) SEC SEC SEC 2"

よしじろうひろもり
5 years ago
Views:

2 SEC (SEC) SEC SEC SEC 2

3 SEC ( 1) 1: SEC 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.

4 ( ) ( 2) ICH (Q6B) 3 lgg Agilent AdvanceBio SEC 3 Å 7.8 x 3 mm 2.7 µm (p/n PL ) Agilent 126 Infinity LC 1. ml/min : IgG min UV 22 nm 5 µl IgG 15 mm ph 7. SEC ( 3) ( ) 3: 4

5 SEC-UV/DAD ( ) mab MW >.1 1,25 kda mab MW >.1 1, kda AdvanceBio SEC (2.7 μm) MW (kda) 13 Å Å 5 1,25 Agilent Bio SEC-5 (5 m) MW (kda) 1 Å Å 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.* ºC 8 C 4.6 mm.1.4 ml/min 7.8 mm ml/min 5 % * Defining the Optimum Parameters for Efficient Size Separations of Proteins ( ) ( EN) ph.2 5 mm ph mm 5 mm ph mm 5 mm ph mm (KCl ) ph mm ph % ( ) 5 mm ph 7. 5 % DMSO SEC 1 3 C SEC Agilent Bio SEC 8 C 5

6 SEC (RI) UV DAD RI 2 M NaCl 8 M ph 1 13 ( ) LC 126 Infinity LC LC Agilent Agilent 126 Infinity LC RFID 1 mm 13 µl (p/n G ) 6

7 HPLC Agilent OpenLAB CDS ChemStation GPC/SEC Agilent GPC/SEC Buffer Advisor ph ph SEC ( ) ( ) ( 4) SEC 17 Mn Mw Mp : SEC Mw Mn Mp 7

com/chem/jp Captiva PES (mm) (μm) 4.45 LC 519-595 4.

8 HPLC HPLC ( ) ( ) ( ) ( ) 5 Captiva PES LC PVDF PES PES LC PVDF Captiva PES (mm) (μm) 4.45 LC LC/MS LC/MS LC LC/MS LC

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.

9 SEC ( ) SEC 7.8 x 3 mm 1. ml/min x 15 mm SEC 2 3 SEC SEC SEC (5 2 µl) (1 4 mg/ ml) ( 7.8 x 3 mm 1. ml/min ) HPLC SEC 2 3 mm 15 mm AdvanceBio SEC 7.8 x 3 mm 1. ml/min IgG 分 AdvanceBio SEC 7.8 x 15 mm 2. ml/min IgG 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

10 ( ) ( 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

11 3 Å 6 5 IgG 3 Å IgG BioRad A: B: A 分 AdvanceBio SEC 3 Å 4.6 x 3 mm 2.7 µm (p/n PL ) AdvanceBio SEC 13 Å 4.6 x 3 mm 2.7 µm (p/n PL ) g- ( ) 3. ( ) 4. ( ) 5. B B Agilent 126 Infinity LC 15 mm ph ml/min UV 22 nm 分 BioRad IgG A 分 A: AdvanceBio SEC 3 Å 4.6 x 3 mm 2.7 µm (p/n PL ) B: AdvanceBio SEC 13 Å 4.6 x 3 mm 2.7 µm (p/n PL ) 1 2, 3 B Agilent 126 Infinity LC 15 mm ph ml/min UV 22 nm 分 IgG 6: BioRad IgG 2 11

12 SEC SEC / / PEG/PEO ( 7) / PEG/PEO / 3 ph 8 5, PEG SEC 5, / 17 (g/mol) 4, 3, 2, 1, 1,, 1, 1, 1, PEG/PEO / (ml) 7: 3 PEG 46,72 ( MV) 48, ( MV) 44,3 ( MV) 分 8: 13 Å AdvanceBio SEC (p/n Å AdvanceBio SEC 2 ml ) 5 ( II) 13 Å Agilent AdvanceBio 3 Å AdvanceBio SEC (p/n Å AdvanceBio SEC 2 ml ) 5 ( g- II) 3 Å Agilent AdvanceBio 12

13 ( / ) 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 ) Bio SEC-5 3 Å 7.8 x 3 mm 5 µm (p/n ) Agilent 126 Infinity LC 15 mm ph 7 1 ml/min UV 22 nm Rs µm 5 µm 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

14 3 mm 15 mm SEC 1 15 mm 2 ml/min 4 IgG A 分 AdvanceBio SEC 3 Å 7.8 x 15 mm 2.7 µm (p/n PL ) 15 mm ph ml/min ( bar) UV 22 nm 5 µl 正規化後 B.5 ml/min 1. ml/min 1.5 ml/min IgG (2 mg/ml) 分 1: 12 4 (A) (B) SEC (1 2 %) ( ) ( ) 5 1 mm Agilent 126 Infinity LC / 11 14

15 (ph) ( 11) A B 5 µl Agilent Bio SEC-3 3 Å 4.6 mm x 3 mm 3 µm (p/n ) Agilent 126 Infinity LC.35 ml/min UV 22 nm A: 2 mm ph mm NaCl B: 2 mm ph mm NaCl C: 2 mm ph mm NaCl (1 mg/ml 2 mm ph 7) 2 mm 5 mm NaCl C mm 4 mm NaCl : 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.

16 ( 12) Agilent Bio SEC x 3 mm 5 µm (p/n ) Agilent 126 Infinity LC 15 mm ph ml/min UV 1,, 1, MW 1 Å 5 Å 3 Å 15 Å 1 Å 67, g- 158, , 1 Å 5 Å BSA 67, , , 3 Å 17, Å A 12, Å (ml) B12 1, : 16

17 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 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) µl 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.

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.

18 / (ADC) SEC ADC AdvanceBio SEC 14 正規化後 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分 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 凝集体インタクト ADC ADC 分解物分 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 LC 4 LC USP 18

19 SEC 25 mm 3 mm mm 1. ml/min 4.6 mm.35 ml/min 15 mm ( ) / / 7 ( ) ( ) ph ( ) SEC 19

20 agilent.com/chem/jp Agilent Technologies, Inc. 215 Printed in Japan November 1, JAJP

GPC/SEC

GPC/SEC GPC/SEC Agilent GPC/SEC...3... 5...5...7...9...11...12... 13...13...14... 15...15...16... 18...18...19 /... 21...21... 23 GPC/SEC...24 Q Q Q Q Q 35 GPC/SEC 24 365 GPC/SEC GPC/SEC GPC 2 80 % 2006 - Polyplasdone