Analysis of IgG1 Free Thiols

Application Notes

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1. Polymer - shell columns for biologics bioVidria, Inc., 1281 Win Hentschel Blvd. , Purdue Research Park, West Lafayette, IN 47906 USA sales@biovidria.com +1 - 855 - 352 - 7690 Application note Resolution of free thiols in IgG 1 by RPLC - MS using formic acid as modifier Authors: Alexis G. Huckabee and Mary J. Wirth , 07/28/2017 Introduction Monoclonal antibodies of IgG 1 are widely used in the pharmaceutical industry, both as drugs and as antibody - drug conjugates. Free t hiols can be introduced by genetic engineering, and the proportion of free - thiol variants can be characterized by RPLC - MS . The challenge is that there are always other variants besides the intended ones, hence the need for mass spectrometry to be used in peak identificatoi n. Conventionally, mobile phase additives, such as TFA and DFA , are used in RPLC - MS. These suppress ionization, hampering peak identification by mass spectrometry. These are avoided here. The unique polymer - shell technology invented by scientists at bioVidria avoids the use of trifluoroacetic acid (TFA), which would otherwise suppress ionization. The technology uses a solid silica core surrounded by a shell of polymer grown from the surface. Th e shell is thick enough to protect the protein from interactions with silanols . The soft polymer surface provide s higher protein recovery. These features enable the most sensitive mass spectrometry in RPLC , as w ell as high protein recovery and LC selectivity . The purpose of this work is to evaluate the performance of the pol ymer - shell technology for resolving IgG 1 thiol variants in a recombinant pharmaceutical sample by RPLC - MS . To avoid ion suppression and adduct formation, no TFA is used in the RPL C - MS. Materials and methods Sample: Pharmaceutical IgG 1 with two free thiols Column: bioVidria IgG RPLC - MS, 2.1 x 50 mm, 1.2 μm UHPLC: Waters Acquity I - Class Mobile phase A: Water, 0.5% formic acid Mobile phase B : ACN, 0.5% formic acid Gradient: 23 - 32% B in 30 min Inj . V ol . : 1 μL in 100% water Column temp: 4 0 °C Sample thermostat: 4 °C Flow rate: 1 00 uL/min @ 350 bar Mass spectrometer: Thermo Velos LTQ Electrospray voltage: +3800 V S heath gas pressure : 40 psi Aux gas pressure: 5 psi Max injection time: 500 ms MS range: 2000 - 3800 m/z Results The c hromatogram for IgG1 with no TFA , using mass spectrometry for detection , is shown to resolve thiols variants , and also to resolve more variants . 5 10 15 20 0 1x10 6 2x10 6 3x10 6 2 free thiols 1 free thiol Total ion current Time (min) no free thiols Conclusions The bioVidria polymer shell column with no TFA provides higher resolution than other commercial columns that use TFA. Better resolution of the extraneous variants facilitates MS identif ication of the thiol variants.

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