The Peptide Mapping Games! Introduction and Manual Sample Prep CSD Tim Rice, Paul Dinsmoor BioColumn Technical Specialists 1
Proteins and Monoclonal Antibodies Complex Molecules, Complex Manufacturing Complex Molecules Proteins have multiple layers of structure primary, secondary, tertiary.., that all must be characterized Monoclonal antibodies/proteins have glycosylations/post-translational modifications that can vary Complex Manufacturing Manufacturing requires living cells which can vary, creating variable results Variation in process can change structures, post-translational modifications These changes need to be characterized 2
Characterization of Primary Structure When to Use reverse-phase Separations 3
Why peptide mapping? Analysis of a single modification on a large protein may not be possible because the change is too small to be seen in the large molecule Results in a collection of peptides of varying length & number depending on the protease used. The resulting peptides can be analyzed where each peak represents a particular peptide treatment with a protease enzyme
Peptide mapping is the single most important technique in analytical characterization Confirm primary structure by comparison of a product to a reference protein (detect point mutations, mis-translations & confirm genetic stability) Identify location of disulfide bonds Characterize & analyze degradation processes such as deamidation & oxidation Isolate digest fragments for sequencing or further identification Identify sites of glycosylation Drug substance identity test Drug substance purity test 5
Analyzing protein drug by RP-HPLC peptide map - pyroglutamate formation Reverse-Phase Chromatography/ Mass Spectrometry Analysis of Reduced Monoclonal Antibodies in Pharmaceuticals Douglas Rehder, Thomas Dillon, Gary Pipes, and Pavel Bondarenko, Journal of ChromatographyA, 1102 (2006), p.164-175 Analyzing protein drug by RP-HPLC Peptide map 1- amino acid substitution Identification of a Glu > Lys substitution in the activation segment of human pepsinogen A-3 and -5 isozymogens by peptide mapping using endoproteinase Lys-C Ruud A. Bank, Bart C. Crusius, Toon Zwiers, Stephan G.M. Meuwissen*, Fre Arwert and Jan C. Pronk Institute of Human Genetics and *Department of Gastroenterology, Free University, Amsterdam. The Netherlands, Volume 238, number 1, 105-108 FEB 06339 September 1988 Analyzing protein drug by RP-HPLC Peptide map-mirror image LC/ESI-MS/MS analysis of recombinant IgG2 mab after Lys-C digest Wypych J et al. J. Biol. Chem. 2008;283:16194-16205 6
Analyzing protein drug by RP-HPLC Peptide map-deamidation Quantification and characterization of antibody deamidation by peptide mapping with mass spectrometry Weijie Wang, Andrea R. Meeler, Luke T. Bergerud, Mark Hesselberg, Michael Byrne, Zhuchun Wu, Analytical Sciences Department, Human Genome Sciences, Inc., 14200 Shady Grove Road, Rockville, MD 20850, United States Analyzing protein drug by RP-HPLC Peptide map - disulfide bonds Disulfide Linkage Analysis of IgG1 using an Agilent 1260 infinity Bio-inert LC System with an Agilent Zorbax RRHD Diphenyl sub-2um Column M. Sundaram Palaniswamy, Agilent Technologies, Inc., Bangalore, India Analyzing protein drug by RP-HPLC Peptide map - PEGylation Toward Top-Down Determination of PEGylation Site Using MALDI In-Source Decay MS Analysis Chul Yoo, Detlev Suckau,Volker Sauerland,Michael Ronk, and Minhui Maa, Analytical R&D, Amgen Inc., Thousand Oaks, California, USA 7
Manual Sample Prep Trypsin is most commonly used protease Digestion is performed optimally at ph 7.5 to 8.5 at 37C 50mM triethyl ammonium bicarbonate (tabc) or 12.mM ammonium bicarbonate (ABC) to achieve ph Tris can also be used, but Tris is not compatible with MS Detailed process can be found in our Peptide Mapping How To Guide part number 5991-2348EN 8
NEW: Peptide Mapping How To Guide #5991-2348EN 9
Peptide mapping of mab Commonly used proteases in producing mab peptide maps Protease Cleavage site Number of mab fragments Trypsin C-terminus of lys & arg 59 Endoprotease Lys-C C-terminus of lys 41 Endoprotease Asp-N N-terminus of asp 30 10
Manual Sample Prep Continued First we denature and reduce our protein by adding TFE and DTT and incubating at 60 C for 45min to 1 hour, then cool to room temp. Next step is to alkylate Iodoacetamide (IAM) is added ( 4ul of stock) Vortex briefly Incubated in dark for 1 hour at room temp Quench excess IAM with DTT, 1 hour in dark at room temp Next, dilute and adjust ph of solution for trypsin digestion 11
Manual Sample prep cont.trypsin Digest Use fresh trypsin stock Add stock solution at 1:20 or 1:50 by mass of enzyme:substrate Incubate 37C for 4-18 hours Cool and then halt trypsin activity by adding 1ul neat formic acid to lower ph below 4 May 5, 2014 12
Peptide mapping example Tryptic digestion of anti-cd4 IgG1 Denature mab Evaporate 5 mg of IgG to dryness and dissolve in 0.5 ml of 6.0 M Gu-HCL, 1.2 M Tris/HCl, 2.5 mm Na 2 EDTA (ph 8.4) buffer Reduce disulfide bonds Alkylate reduced cysteine Buffer exchange Reduce disulfides by adding 25 ul of 1.0 M DTT (40 folds excess) and placing at 65ºC for 30 min S-carboxymethylate by adding 60 ul of 1.0 M sodium iodoacetate (1.2 fold molar excess) and placing at room temperature, in the dark for 40 min. Halt alkylation by adding 15uL of 1.0 M DTT Desalt and exchange into digestion buffer by applying reaction mixture to Bio-Gel P-6DG gel column equilibrated with 50 mm Tris/HCl, 1 mm CaCl 2 (ph 8.1)digestion buffer Elute and collect the carboxymethylated protein in 2.2 ml of digestion buffer Protease digestion Digest one 500 ul portion of carboxymethylated protein (2 mg/ml) by adding 10 ul of 1.0 mg/ml trypsin and placing at 37ºC for 2 h Halt digestion by adding 25 ul of 1.0 M to give ph 2. Save rest of S-carboxymethlated protein at -70ºC RP-HPLC or LC-MS 13 May 5, 2014
Sample Prep Some analyses require a digestion clean up step Depending on sample origin, or if you used TFA instead of formic acid, you may need to desalt prior to MS analysis Use Agilent Bond Elut OMIX C-4 tips pipette tips as described in application note # 5990-8885EN If desalting is not necessary, but sample appears opaque, filter the sample prior to MS Use Agilent Spin Filters, p/n 5185-5990 May 5, 2014 14
May 5, 2014 15
Thank you! QUESTIONS? 16