ISPPP 2017 Poster

Presentations

Share on Social Networks

Share Link

Use permanent link to share in social media

Share with a friend

Please login to send this presentation by email!

Embed in your website

Select page to start with

Post comment with email address (confirmation of email is required in order to publish comment on website) or please login to post comment

1. High Resolution Chromatography Coupled with Mass Spectrometry for Therapeutic IgG1, IgG2, and IgG4 www.biovidria.com sales@biovidria.com Phone: 855 - 352 - 7690 Objectives Polymer - shell technology Mary Wirth 1,2 , Alexis Huckabee 1 , Yiyang Zhou 2 and Jonathan Yasosky 2 1 bioVidria, Inc., 1281 Win Hentschel Blvd, West Lafayette, IN 47906 2 Purdue University, Department of Chemistry, West Lafayette, IN 47907 • Screens surface charges to reduce tailing • Sterically prevents protein - silica contact • Envelops protein to increase selectivity 1.2  m 20 nm LCMS of monoclonal antibodies, ADCs, and other protein drugs requires new ideas in column design rather than adapting columns from the realm of small - molecule drugs. The design investigated is a core of solid silica and a shell of dense polymer brush for attaining high resolution in liquid chromatography and high sensitivity in mass spectrometry for protein drugs. The design is tested for its ability to separate without trifluoroacetic acid (TFA) since TFA suppresses ionization and forms ion pairs with proteins. This is especially a problem with mAbs and ADCs. The polymer layer provides a distance between silanols and the proteins to avoid tailing. The polymer contains multiple functional groups that introduce more selectivity . The design is demonstrated here to give unprecedented LC resolution and MS sensitivity in RPLC of IgG1 and IgG2. Comparable large advantages are obtained for IgG4 and reduced ADCs. The design is being extended to HIC , HILIC and IEX. 0 5 10 15 20 0 200 400 600 800 1000 1200 Pressure (bar) Time (min) Low back - pressure avoids frictional heating Unique benefits for proteins Only 0.082 W at optimal flow rate of 0.2 mL/min Core - shell, Diphenyl bonded phase Hongbin Liu, et al. J. Pharm. Biomed. Anal. 109 (2015) 142 – 149 RPLC - MS of IgG1 Time (min) 0.1% FA + 0.025% TFA 6% ACN/min 10 mAU 1. Comparison with published results shows that resolution for free thiols is higher for the polymer shell column using MS - compatible mobile phase. 2 . Study of both MS sensitivity and LC resolution reveals that 0.25% FA+0.025%DFA gives best LC resolution and best MS sensitivity: P = 245 to 247 bar during separation 0.1%FA+0.025%TFA 18 20 22 24 26 28 0.00 0.02 0.04 0.06 AU (280 nm) 18 20 22 24 26 28 0 1x10 6 2x10 6 3x10 6 4x10 6 MS TIC Time (min) 0.25%FA+0.025%DFA 22 24 26 28 30 32 0.00 0.02 0.04 0.06 AU (280 nm) New shoulders 22 24 26 28 30 32 0 1x10 6 2x10 6 3x10 6 4x10 6 MS TIC Time (min) UV : MS: 0.1%DFA 0.5%FA 14 16 18 20 22 24 0 1E6 2E6 3E6 4E6 MS TIC Time (min) 12 14 16 18 20 22 0 1x10 6 2x10 6 3x10 6 4x10 6 MS TIC Time (min) 12 14 16 18 20 22 0.00 0.02 0.04 0.06 AU (280 nm) 14 16 18 20 22 24 0.00 0.02 0.04 0.06 AU (280 nm) • New peaks are resolved by polymer shell column for the same Genentech IgG1. Polymer - shell mAbs courtesy of Pfizer, Genentech Highest Ion count Resolution of disulfide isoforms in IgG2 is higher for polymer shell column, and MS - compatible mobile phase gives high resolution. 0 5 10 15 20 Time (min) Gradient was 4% ACN/20 min, with varying starting point. • Peaks are further apart for the same gradient, showing the higher selectivity. 0 5 10 15 20 Time (min) a) Polymer shell gives higher resolution than core - shell for Pfizer IgG2. Core - shell P olymer - shell b ) MS - compatible mobile phase gives little sacrifice in resolution. RPLC - MS of IgG2 4% ACN/20 min 0.1% TFA 4% ACN/20 min 0.1% TFA Silica core NIST mAb 2.1 mm x 50 mm 0 5 10 15 20 0.000 0.001 0.002 0.003 AU (280 nm) Time (min) 4% ACN/20 min 0.1% FA+ 0.025% DFA P olymer - shell

Views

  • 56 Total Views
  • 31 Website Views
  • 25 Embeded Views

Actions

  • 0 Social Shares
  • 0 Likes
  • 0 Dislikes
  • 0 Comments

Share count

  • 0 Facebook
  • 0 Twitter
  • 0 LinkedIn
  • 0 Google+