Characterizing Deamidation and Oxidation in Adalimumab with Low pH Peptide Mapping and Middle-Up Mass Spec Analysis Chris Hosfield1, Eric Largy2, Anicet Catrain2, Fabrice Cantais2, Géry Van Vyncht2, Michael Rosenblatt1, Sergei Saveliev1, Marjeta Urh1 & Arnaud Delobel2 1Promega Corporation, Madison, WI, USA, 2Quality Assistance, Thuin, Belgium 1. Introduction 3. Analysis of Adalimumab Deamidation 4. Analysis of Adalimumab Oxidation A. Comparison of Conventional and Low-pH Digestion A. Oxidation Study of Adalimumab using IdeZ Digestion and Middle-Up MS Non-enzymatic chemical modifications such as deamidation, oxidation and disulfide bond scrambling can affect the stability and efficacy of biotherapeutic proteins. Peptide mapping, the method of choice for site-specific monitoring of these modifications, has a significant drawback Fc/2 F(ab’ )2 which is that the sample preparation methods often induce the same modifications intended to Ox Non oxidized Ox x 2 Ox be measured. A primary source of these sample preparation artifacts is the alkaline pH favored Ox x 2 Ox Ox Non oxidized by trypsin and other commonly used proteases. To address this problem we have developed a 0.2 % Raw-MS Fc/2 Spectra Deconvoluted Fc/2 Spectra sample preparation procedure in which all steps including proteolytic digestion are performed under acidic conditions. Although trypsin is inhibited at low pH, we have overcome this limitation by supplementing the digestion with a low pH resistant Lys-C protease. Here we describe the Base Peak Chromatogram – Conventional pH Digest Base Peak Chromatogram – Low pH Digest utility of this new sample preparation procedure for analysis of deamidation in Adalimumab H2O2 ↗ (Humira). We show that observed deamidation sites appear to be artifacts induced during RT ↗ conventional sample preparation procedures. In addition, we have analyzed peroxide-induced 100% 100% oxidation in Adalimumab, both by peptide mapping and subunit-level analysis following digestion 75% 75% Oxidation with IdeZ protease. An oxidation suppressant added to the peptide mapping protocol reduces 0 % oxidation when digestion occurs in the presence of peroxide. 50% 50% Alkaline pH Digest 25% 25% Low pH Digest H2O2-induced oxidation of Fc and F(ab’)2 Middle-up MS analysis of Fc and oxidized Fc fragments Modification Modification Level Sequence coverage 0% Alkaline pH Low pH Digest 0% 2. Study Design Digest B. Addition of an Oxidation Suppressant Reduces Peroxide-Induced Met-Oxidation Digestion at low pH produces Digestion at low pH does not affect C-terminal HC:M256ox HC:M83ox HC:M34ox Deamidation Analysis: Low pH vs Conventional Digestion equivalent sequence coverage Lysine truncation, glycosylation or pyroglutamate. Deamidation Artifacts No suppressant B. XIC Analysis of “PENNY” Peptide Deamidation (GFYPSDIAVEWESNGQPENNYK) LC-MS Conventional Low pH Digestion Digestion Suppressant LC-MS Base peak comparison of digestion in H2O2 with or without an oxidation suppressant Adalimumab was denatured, reduced, alkylated and digested into peptides using either a conventional 100.0 protocol (pH 8) with Trypsin or with a low pH protocol utilizing Trypsin and a low-pH resistant LysC. DeAm 80.0 Native DeAm DeAm Native DeAm 60.0 Succinimide Int. 40.0 S- No suppressant Oxidation (%) Oxidation Oxidation Analysis: Suppression of Peroxide-Induced Oxidation - S+ Suppressant Succinimide Int. Met 20.0 0.0 LC-MS ~6% deamidation observed at conventional pH ~0.5% deamidation observed at low pH Methionine oxidation can be reduced with an oxidation suppressant added during digestion Low pH Digestion Kit Oxidation Sites C. Deamidation Summary for all Peptides LC-MS and Data Analysis: IdeZ-digested Adalimumab fragments were separated on a MAbPac RP column (3.0 LC-MS 12.0 x 50 mm, 4 µm; Thermo Scientific) heated to 80°C. UV detection was set at 280 nm, and MS detection at 500- 10.0 4000 m/z. Deconvolution was achieved using the MaxEnt1 algorithm. Tryptic digests analyzed as previously 8.0 described. Oxidation was confirmed by MS/MS and loss of methyl sulfoxide on all fragments that contain MetOx. Adalimumab was exposed to peroxide in the presence or absence of an oxidation suppressant while 6.0 simultaneously being digested at low pH with Trypsin and low-pH resistant LysC. 4.0 % Deamidation 2.0 5. Conclusions Oxidation Analysis: Middle-Up MS with IdeZ Protease 0.0 2:T24 1:T21-22 2:T35 2:T13 2:T8 2:T34 2:T5 2:T21 We have developed a method for peptide mapping at low pH Low pH digestion reduces deamidation in all peptides. Key Benefits of this method: H2O2 IdeZ LC-MS LC-MS and Data Analysis: Digests were separated on an H-Class UPLC (Waters) with a CSH130 C18 1.7 µm, • Robust digestion: Sequence coverage is equivalent to conventional pH digestion. 2.1 x 150 mm column (Waters) with UV (214 nm) and Xevo G2-XS mass spectrometer detection (Waters). Data • Artifact suppression: Deamidation and disulfide bond scrambling are not introduced. were processed with UNIFI 1.7 (Waters) with a 10 ppm m/z tolerance and 1 missed cleavage allowed. Oxidation Sites • Oxidation during sample prep can be reduced with an optional oxidation suppressant. Adalimumab was oxidized with increasing peroxide concentrations for 45 min, then digested with IdeZ Low pH digestion kit efficiency is equivalent to conventional methods protease for 30 min into F(ab’)2 and Fc fragments. Middle-up MS Analysis was performed to monitor oxidation. and is superior for deamidation analysis June 2016 www.promega.com Corresponding author: [email protected].