Direct LCMS analysis method of surfactants contained in

Direct LC/MS analysis method of surfactants contained in antibody drugs with using polymer-based reversed phase column PITTCON 2019 March 17 – 21, Philadelphia Shodex Junji Sasuga 2, Hiroki Takenaka 2, Hirobumi Aoki 2, Eiji Kagawa 2, Leah Sullivan 1, and Ronald Benson 1 CAPTURE THE ESSENCE 1 Showa Denko America, Inc. , 420 Lexington Avenue, Suite 2335 A, New York, NY 10170, USA, 2 Showa Denko K. K. , 5 -1 Ohgimachi Kawasaki-ku, Kawasaki, 210 -0867, Japan Experimental Introduction Ig. G In order to prevent ion suppression by proteins, salts, strain on MS, after these are eluted, the eluent was introduced into MS ODS Polyhydroxymethacrylate Silica Functional group None Octadecyl (C 18) Average pore size 40Å 100 - 120Å Base material Polymer-based Available p. H 3 – 12 Sample: Tween 20 47 mg/L + Ig. G 1 g/L + Na. Cl 0. 3 mol/L (in H 2 O) 2 - 8 Ig. G and inorganic salt were eluted at V 0 UV 280 nm Proteins are easily adsorbed, allowing for the retention of hydrophilic molecules strong log k' 1. 00 Retention ODP 2 HP-4 D 0. 00 ODS 1 weak -0. 50 ODS 2 -1. 00 -1 High 0 log P Polarity 1 2 Low Columns: Shodex ODP 2 HP-4 D Column ODSs from two other manufacturers 4. 6 mm I. D. x 150 mm each Eluent: H 2 O / CH 3 CN = 75 / 25 Flow rate: 1. 0 m. L/min Column temp. : 40 o. C Compared to the ODS, ODP 2 HP has higher polarity of the packing material and is more likely to retain a polar compound 10 15 20 SIM m/z 735. 7(+) 5 0 10 15 20 min ODS UV 280 nm 20 40 60 80 100 120 140 UV 280 nm into MS Number of BSA injection This component was monitored Recovery rate 107% SIM m/z 848. 8(+) 0 5 10 ・When alkaline eluent was used, Ig. G became more hydrophilic, and was not retained in the column　 into MS Sample: 7 mg/m. L BSA (in H 2 O), 5 μL Columns: Shodex ODP 2 HP-2 B Column : ODS from other manufacturer 2. 0 mm I. D. x 50 mm each Eluent: 1 m. M CH 3 COONH 4 aq. / CH 3 CN = 90 / 10 Flow rate: 0. 2 m. L/min Column temp. : 30 o. C ODP 2 HP showed stable pressure even after 140 injections, indicated no BSA retention assumed to be a divalent ion consisting a sorbital backbone, 20 or more EO units, glycerol diesters (MW 1435) of C 14 and C 12 with either one having a double bond, and two ammonium ions assumed to be a divalent ion consisting a sorbital backbone, 28 or more EO units, glycerol monoester (MW 1661) of C 18 with a double bond, and two ammonium ions Ig. G and inorganic salt were eluted at V 0 ODP 2 HP-2 B 15 20 min 0 ・Tween was not subjected to ion suppression even when sample containing Ig. G and Na. Cl were analyzed SIM m/z 848. 8(+) 5 10 15 20 2 3 4 5 6 7 8 9 min cf. The concentration published from the manufacturer: 7. 0 mg / 10 m. L ・It was confirmed that Tween 80 can be accurately quantified ・Using the 50 mm column, analysis time can be shortened to 10 min (including equilibration time) <m/z 848. 8(+)> (b) with Ig. G 1 g/L Ig. G + Na. Cl 0. 3 mol/L 120 90 Tween 20: R = H or C 11 H 23 CO Tween 80: R = H or C 17 H 33 CO w + x + y + z ≈ 20 <m/z 735. 7(+)> min 0 (a) without Ig. G + Na. Cl 140 100 Sample: Commercial antibody drug 10*dil (in H 2 O), 2 μL Column: Shodex ODP 2 HP-2 B (2. 0 mm I. D. x 50 mm) Eluent: (A) 0. 1% NH 3 aq. / (B) CH 3 CN High pressure linear gradient B% = 20% (0 min) 90% (5 -6 min) 20% (6. 1 -10 min) Flow rate: 0. 2 m. L/min Column temp. : 40℃ Detector: PDA (190 -350 nm) & ESI-MS(+) Scan / SIM 1 Quantitative result of Tween 80: 702 μg/m. L Sample: Tween 80 57 mg/L + Ig. G 1 g/L + Na. Cl 0. 3 mol/L (in H 2 O) 110 ・The most abundant component was selected by pre-scanning and the calibration curve was prepared from the results by SIM of the ion Recovery rate 97% SIM m/z 735. 7(+) 5 0 Model sample containing Tween 80 160 130 SIM m/z 848. 8(+) ・Tween is not a single structure, but a complex mixture containing components of various structures into MS This component was monitored and the calibration curve of Tween 20 was prepared 0 Absorptivity of protein % Relative column back pressure ( observed P / original P) Polarity of packing material UV 280 nm into MS ・Housing： SUS ・Available temp. : 20～ 60℃ ・Available solv. ： H 2 O, CH 3 CN, CH 3 OH 0. 50 (b) with Ig. G 1 g/L + Na. Cl 0. 3 mol/L (a) without Ig. G + Na. Cl Ig. G Scan TIC m/z 500 -2000(+) Model sample containing Tween 20 Small hydrophilic molecule ODP 2 HP-2 D Tween 80 Results and Discussion Alkaline available Both hydrophobic and hydrophilic molecules are retained while proteins are not into MS Quantification of Tween 80 by monitoring this peak Small hydrophobic molecule ODP 2 HP-2 B UV 280 nm Sample: Tween 20 or 80 + Ig. G + Na. Cl (in H 2 O), 2 μL Instrument: Shimadzu Nexera / LCMS-8030 Plus Column: Shodex ODP 2 HP-2 D (2. 0 mm I. D. x 150 mm) Eluent: (A) 0. 1% NH 3 aq. / (B) CH 3 CN High pressure linear gradient B% = 20% (0 -5 min) 90% (19 -24 min) 20% (25 -30 min) Flow rate: 0. 2 m. L/min Column temp. : 40℃ Detector: PDA (190 -350 nm) & ESI-MS(+) Scan / SIM Shodex. TM ODP 2 HP Application to actual sample Commercial antibody drug containing Tween 80 During the production of antibody drugs, small amounts of surfactants may be used to solubilize or stabilize Ig. G. The determination of the surfactant concentration is an important QC factor. The quantification of surfactants can be difficult as they exist within complex matrices and have low UV absorption. Through the use of a polymer-based reversed phase column, Shodex. TM ODP 2 HP series, we developed a rapid LC/MS method for quantifying polysorbate (Tween) in the presence of Ig. G and Na. Cl, without the need to deproteinization nor desalt sample pretreatment. TM min ・Highly sensitive analysis of Tween can be efficiently performed without preprocessing for desalting and the removal of proteins Conclusions Shodex™ ODP 2 HP-2 D coupled with MS proved its separation capability of analyzing non-ionic surfactant, Tween, in the presence of Ig. G and Na. Cl without the necessity of sample pretreatment, deproteinization, or desalting the sample. Here, alkaline eluent was effective for eluting Ig. G at V 0, which can was completed using a polymer-based reverse phase column, such as ODP 2 HP, that is durable against alkaline conditions. The method developed in this application is fast and high-sensitive for the analysis of Tween which has potentials in antibody drug QC analysis. Faster analysis can be achieved by using a shorter column (50 mm), ODP 2 HP-2 B. 　 Contact us at support@shodex. HPLC. com For more information, visit www. shodex. HPLC. com