크기 크로마토그래피(SEC)를 분석 - Agilent

1 (SEC) 2 SEC (SEC: Size Exclusion Chromatography) . , SEC . SEC ( (aggregates), (Excipients), (cell debris) ) (intact proteins) . SEC . SEC , , , , SEC .3 1: pore pore . pore .SEC . (void volume) . pore ( 1), pore . SEC .4 , . (dimers, trimers, tetramers ) ( 2).

2 , . ICH(Q6B) .. SEC .. , (hydrodynamic radius) ( 3). , ( ) . 3: 2: IgG : Agilent AdvanceBio SEC, 300 x 300mm, m (p/n PL1180-5301) : Agilent 1260 infinity Bio-inert quaternary LC : : : UV, 220nm : 5 L : IgG : 150mM , pH lgG Monomer Dimer min2468101214mAU01020304050607080123451. 2. (Dimer)3. (Monomer)4. Fragments5. (Excipients)5 SEC-UV/DAD , , , , , , mAbsMW > ,250kDa pore , , , mAbsMW > ,000kDa.

3 PH . , .. , :100-150mM 50mM (pH ) 100-150mM 50mM (pH )50-100mM (urea) 50mM (pH ) ( : KCl) (guanidine hydrochloride) .pH : :5-10% ( ) 50mM (pH ), 5% DMSO Agilent Bio SEC-5(5 m)pore (kDa)100 0150 -15 0300 5 -1, 25 0500 15-5,0001000 50-7,5002000 >10,000 AdvanceBio SEC( m)pore (kDa)130 0300 5 -1, 25 050mM (pH ). , . : , SEC 10-30 C .. SEC . Agilent Bio SEC 80 C .. : (Defining the Optimum Parameters for Efficient Size Separations of Proteins)( 5990-8895EN) : AdvanceBio SEC Agilent Bio SEC-5 : 150mM , pH * : 10-30 : : 10-30 C, : 80 C : id id : 5% * 6 Agilent 1260 infinity Bio-inert quaternary LC SEC.

4 , (RI) UV DAD . (baseline) , RI .. 2M NaCl 8M (Urea) pH (1~13) , LC . 1260 infinity Bio-inert quaternary LC . , . , . LC . (autosampler) ..SEC RFID , 10mm, 13 L(p/n G5615-60022)7 4: SEC Mw, Mn Mp . HPLC : Agilent OpenLAB CDS ChemStation.

5 GPC/SEC : Agilent GPC/SEC , . Buffer Advisor : pH , pH scouting . SEC ( , ) ( ) ( 4). , .. 17 . MpMnMw8 Captiva , . Agilent PES . LC , PVDF PES . Agilent PES LC PVDF , PVDF .. HPLC .. SEC HPLC ( ).

6 , . stress (dimers multimers ) ( sub-units ) . - , , . 5 .Captiva PES (mm)pore ( m) 5190-50999 SEC , . SEC x 300mm , x 150mm SEC 2-3 . SEC . SEC . SEC (1-4mg/mL) (5-20 L) . 10-12 ( x 300mm mL/ ), . HPLC , SEC .. pore . , pore.

7 , pore . 300mm .. 150mm , . , (backpressure) , ( 5 ). 5: 300mm 150mm : AdvanceBio SEC, x 300mm : : IgG : AdvanceBio SEC, x 150mm : : IgG 14 1. 2. (Dimer)3. (Monomer)4. Fragments5. (Excipients) 10 ( , ) , . ( , ) . ( 1). 1: Agilent Bio SEC Agilent GPC.

8 ( , , / ) .. , . (SEC) mAbs, SEC-UV/DAD LS Agilent AdvanceBio SEC .mAbs, SEC-MS Agilent Bio SEC-3MS (baseline) . Agilent Bio SEC-5 Pore (100 , 150 , 300 , 500 , 1000 , 2000 ) . , ProSEC 300S . , ZORBAX GF-250/450 USP L35 . , , PEG, 2 3 PL aquagel-OH PL aquagel-OH 8 m PL aquagel-OH 20 5 m PL aquagel-OH MIXED-M 8 mPL aquagel-OH pH 2-10 ( 50% ) , 140bar(2030psi) . , , 2 3 PL aquagel-OH PL aquagel-OH MIXED-H 8 m PL aquagel-OH 60/50/40 8 m , , , (gums)PL aquagel-OH 60/50/40 15 m 11min1 2, 3 4 5 mAU24681012140204060801001 2 3 4 5 2468101214minmAU010203040506070minmAU246 810121401002003004005006001 2 3 4 5 pore pore 300.

9 6 pore 5 IgG , BioRad 6: BioRad IgG pore . pore . pore pore . 300 pore IgG dimer . pore .1. 2. (Dimer) 3. (Monomer) 4. Fragments 5. (Excipients)min2468101214mAU025507510012 51501752001 2 4 5 3 A: AdvanceBio SEC 300 x 300mm, m(p/n PL1580-5301) B: AdvanceBio SEC 130 x 300mm, m(p/n PL1580-5350) A: AdvanceBio SEC 300 x 300mm, m(p/n PL1580-5301) B: AdvanceBio SEC 130 x 300mm, m(p/n PL1580-5350) : Agilent 1260 infinity Bio-inert quaternary LC : 150mM , pH : : UV, 220nm : BioRad : Agilent 1260 infinity Bio-inert quaternary LC : 150mM , pH : : UV, 220nm : IgG IgG 1.

10 2. g- ( ) 3. ( ) 4. ( ) 5. B12 B A B A124,000mAU3,0002,0001,0000246810min1001 ,00010,000100,0001,000,00045678910111213 SEC , SEC . 7 , / (pullulan)/ PEG/PEO , . (pullulan)/ PEG/PEO calibrant , / . 3 . pH .. , 50,000 calibrant . ( 8). PEG 7 , . SEC . , calibrant . , 50,000 (pullulan)/ . 16 . 7: calibrant 8: calibrants / PEG/PEO (g/mol) (mL) , 44,300 ( ) 48,000( )PEG 46,702( )130 AdvanceBio SEC (p/n 5190-9416 130 AdvanceBio SEC , 2mL ) Agilent 130 AdvanceBio 5 ( , , , , II).