Monoclonal Antibody Characterization Achieving Higher Throughput and Productivity

Monoclonal Antibody Characterization Achieving Higher Throughput and Productivity

Dionex Solutions to Accelerate Monoclonal Antibody R&D and Characterization The throughput and productivity challenge Increasing number of MAb candidates entering the clinical pipeline Advances in automation in upstream processes like cell culture and purification process development, and formulation screening Strict Quality-by-Design (QbD) guideline requiring enhanced antibody characterization All result in a large increase in sample requests for characterization. Drug Discovery Target Identification and Validation MAb Generation Preclinical Development Cell Line Development Clone Screening and Selection Clinical Development Cell Culture and Purification Process Development Pre-Commercialization Formulation Lot Release Testing Stability Studies Analytics Purity Aggregates Charge Variants Peptide Mapping Glycans Post-Commercialization Product Improvements, Patent Extensions, Biobetters The analytical solutions Dionex provides best-in-class analytical solutions for MAb therapeutics, improving throughput and productivity, thus reducing time to market. Best-in-class columns for high-resolution and high-throughput characterization using ion-exchange chromatography (IEC) and size-exclusion chromatography (SEC) MAb characterization platforms to increase sample throughput and streamline multistep workflows Powerful and flexible Chromeleon Chromatography Data System software Innovative UltiMate 3 RSLC system for UHPLC solutions for fast MAb characterization and peptide mapping High performance MAb glycan analysis 2

Whatever Your Workflow, You Can Achieve Higher Throughput and Productivity Multistep MAb characterization workflows MAb clone selection workflow Samples from Cell Line Development MAb Capture by Protein A Aggregate Analysis by SEC Charge Variant Analysis by IEC Clone Selection MAb aggregate or charge variant characterization by LC/MS MAb Samples Separation by IEC or SEC Fraction Collection Desalting on RP C18 Column MS Analysis High-throughput MAb characterization workflows High-throughput MAb titer assay using parallel or dual Protein A column setup Titer Assay Protein A (Column 1) Samples from Cell Line Development Titer Assay Protein A (Column 2) Selection of High-Titer Producing Clones High-throughput MAb aggregate and charge variant characterization using parallel setup SEC or IEC Column 1 Samples from Process Purification or Formulation Data back to Process Purification or Formulation SEC or IEC Column 2 Dual method MAb aggregate and charge variant characterization using parallel setup Assay 1 Aggregates by SEC Samples from Process Purification or Formulation Assay 2 Charge Variants by IEC Data back to Process Purification or Formulation 3

The Dionex MAb Characterization Platform UltiMate 3 MAb fully biocompatible platform Titanium pumps; PEEK fluidics, valves and injection needle Full compatibility with all biological buffers Prevents iron poisoning of columns Maintains protein modifications Less system corrosion and maintenance Dual gradient pump design: 2-in-1 system Multistep Automation: allows automation of two or more methods like Protein A capture, SEC aggregate, and IEC charge variant analysis Tandem Analyses: shortens run times by utilizing the power of off-line column regeneration Parallel LC: double throughput for productivity, cost, and space saving UltiMate 3 MAb Analysis Platform with dual ternary gradient pumps and autosampler with integrated fraction collector. Autosampler with fraction collection and re-injection Dual valve design allows injection, fraction collection, and re-injection Optimized for automated workflows like: Automated multistep workflow automation Protein purification Sample fractionation and desalting prior to MS detection Sample derivatization like digestion or neutralization between multistep separations 4

For Your Throughput and Productivity Needs Chromeleon Chromatography Data System (CDS) software Features of Chromeleon CDS software: Automated peak integration Automated control of fraction collection into autosampler Automated method template and sequence generation for multi-dimensional workflows Automatic rejection of samples based on set criteria e.g., automated rejection of clones with low antibody titer Dilutions or variable injection volumes for the next steps One-click report generation Validation report templates and sequences for increased automation of method validation Fully integrated solutions for R&D, analytical method development and QA/QC. 5

Boost Productivity with Automated Multistep MAb Capture and Characterization Automate your multistep MAb capture and characterization reduce hands-on time and increase productivity Dual Ternary Gradient Pump. 2-in-1 Pump Design. Step 1: Protein A capture of IgG (MAb) and automated fraction collection. 4 Purified Antibody -5 1 2 3 4 5 Step 2: SEC aggregate analysis. Step 3: IEC charge variant analysis. 2 35 Lysine Variants MAb Monomer K Aggregates Acidic Variants KK Basic Variants Autosampler with Injection, Fraction Collection, and Re-Injection. Two sampling technologies in one. 5 1 15 2 25-5 1 2 3 4 5 6 7 8 9 The Multistep MAb Analysis Platform allows the purification and analysis of hundreds of MAb samples. Cell culture fluid samples containing antibodies are injected onto a Protein A column for antibody recovery, then the purified antibody samples are automatically injected, first onto the SEC column, and then onto the IEC column fully automated. Pump 1 Pump 2 Waste SCX TCC L R Protein A SEC UV OUT IN The diagram shows the flowpath configuration of the Protein A, SEC and IEC columns for fully automated MAb capture followed by size and charge characterization. 6

Accelerate Product Development by Increasing Sample Throughput Analyze over a 1 MAb samples a day by utilizing the power of off-line column regeneration Column 2 Dual-Gradient Pump Analysis Flow Autosampler Waste Off-line Column Regeneration Flow Column 1 Detector Without Alternating Regeneration Analysis 1 Regen. Analysis 2 Regen. Analysis 3 With Alternating Regeneration Analysis 1 Regen. Analysis 3 Analysis 2 Regen. With the same samples perform multiple separation steps including SEC and IEC in a parallel configuration 2 MAb Monomer Dual-Gradient Pump From Right Pump Column 1 Detector Aggregates 5 1 15 2 25 From Left Pump Column 2 Detector 35 Lysine Variants Autosampler K KK Acidic Variants Basic Variants -5 1 2 3 4 5 6 7 8 9 7

Monoclonal Antibody Characterization Using Charge Characterization with IEC Glycosylation Sialylation C-Terminal Lysine Deamidation Glutamine cyclization Maleuric acid adduct Oxidation Cysteinylation Disulfide related Succinimide Isomerization Column ProPac WCX-1 (4 25 mm) ProPac WCX-1HT (4 5 mm) NEW! MAbPac SCX-1 (4 25 mm) MAbPac SCX-1 (4 15 mm) MAbPac SCX-1HT (4 5 mm) MAbPac SCX-1 for LC/MS (2 25 mm) Hydrophobicity Characterization with HIC Isomerization Succinimide Oxidation Amidation Aggregation Clipping Column ProPac HIC (4.6 25 mm) ProPac HIC (4.6 1 mm) ProPac HIC-1 (2.1 1 mm) ProPac HIC-1 (7.8 75 mm) Size Characterization with SEC Monomers, aggregates, and fragments Under non-denaturing conditions using both high- and low-salt mobile phases and volatile eluents for LC/MS Column MAbPac SEC-1, 5 µm, 3 Å (4. 3 mm) MAbPac SEC-1, 5 µm, 3 Å (4. 15 mm) 8

High-Resolution Columns A 6 B Lysine Variants 8 K 11 KK Acidic Variants K KK Basic Variants 5 4 1 2 3 7 9 1 12 13 14 15 16 17 1 2 3 4 5 58 27562 A. MAbPac SCX-1 column is the next generation IEC column for MAb charge characterization from Dionex. B. ProPac WCX-1 column (inset) is the industry gold standard for charge variant characterization. 8 Main Peak Methionine Oxidation 5 1 15 2 25 3 2841 ProPac HIC-1 column - Separation of populations of MAb variants using hydrophobic interaction chromatography (e.g., methionine oxidation monitoring). 15 A 2 2 B MAb 2 2. 1.43%. 5 6 7 8 9 1 MAbPac SEC-1 MAb Competitor T 3 6 9 12 15 27619 A. MAb analysis using the MAbPac SEC-1 column demonstrating excellent ruggedness. 3 6 9 12 15 B. MAb analysis in volatile buffer for LC/MS MAb Pac SEC-1 vs the competitor s column. 27618 9

Fast MAb Characterization and Peptide Mapping Method acceleration using the new MAbPac SCX-1 column, 4 15 mm 1 Gradient time: 3 min Total analysis time: 45 min -1 46. 2. 18-2 46. 2. Gradient time: 15 min Total analysis time: 3 min 22-2 46. 24. Gradient time: 7 min Total analysis time: 15 min 5 1 15 2 25 3 35 4 45 2842 Elution profiles of a MAb sample from a 4 15 mm MAbPac SCX-1 column with different gradient elution times. The gradient time and total analysis time is indicated in the chromatogram. Fast MAb peptide mapping application of UHPLC and reduced particle size 175-1 13 5 µm -1 12 3 µm -1 2 µm 1 2 3 4 5 6 7 8 9 1 11 12 2843 Performing peptide mapping with smaller particle columns allows the user to achieve identical resolution in less time. Here a method transfer was performed using 2.1 1 mm columns packed with 5, 3, and 2 µm particles respectively on the UltiMate 3 Rapid Separation LC (RSLC). 1

The Preferred and Proven Platform for MAb Glycan Characterization MAb glycosylation why measure it? Increasing relevance biosimilars Glycosylation can affect: Biological activity Pharmacokinetics and clearance in vivo Stability Immunogenicity Analysis of glycosylation is important to: Meet regulatory requirement Ensure product consistency Develop new generation drugs with modified glycosylation Dionex solution High Performance Anion-Exchange with Pulsed Amperometric Detection (HPAE-PAD) The workhorse in carbohydrate analysis High Sensitivity.1 to 1 pmole detection limits Label-Free No derivatization necessary Column: CarboPac PA2 ( 3 15 mm) +/- AminoTrap (3 3 mm) Eluent: 12 mm NaOH Flow Rate:.5 ml/min Detection: Pulsed electrochemical, Au electrode Waveform: Quadruple potential 8 nc 1 2 3 4 5 6 With AminoTrap No AminoTrap Peaks: 1. Fucose 2. Galactosamine 3. Glucosamine 4. Galactose 5. Glucose 6. Mannose -5 5 1 15 2 25 3 Protein hydrolysate Mix of 6 standard Protein hydrolysate Mix of 6 standard 18778 Profiling MAb hydrolysate on the CarboPac PA2 column, with and without an AminoTrap precolumn. 145 Column: CarboPac PA2 and guard Eluent: 5 min: 1 mm NaOH/5 mm NaOAc 6 min: 1 mm NaOH/18 mm NaOAc Temperature: 3 C Flow Rate:.5 ml/min Inj. Volume: 9 µl from 1 µl loop Detection: PAD (Au) Waveform A (TN 21) Samples: A) PNGase F digest of human lgg B) Sialic acids standard C) Neuraminidase digest of Sample A Neu5Ac Neu5Gc nc C B 15 5 1 15 2 25 A 3 2552 ICS-5 New capillary system for carbohydrate analysis. Monitoring release of sialic acids from human lgg N-linked oligosaccharides by HPAE-PAD. 11

Selected Peer Reviewed Publications 1. Lyubarskaya, Y.; Houde, D.; Woodard, J. Murphy, D.; Mhatre, R. Analysis of Recombinant Monoclonal Antibody Isoforms by Electrospray Ionization Mass Spectrometry as a Strategy for Streamlining Characterization of Recombinant Monoclonal Antibody Charge Heterogeneity. Anal. Biochem. 26, 348, 24 39. 2. Vlasak, J.; Ionescu, R. Heterogeneity of Monoclonal Antibodies Revealed by Charge-Sensitive Methods. Curr. Pharm. Biotechnol. 28, 9, 468 481. 3. Valliere-Douglass, J.; Wallace, A.; Balland, A. Separation of Populations of Antibody Variants by Fine Tuning of Hydrophobic Interaction Chromatography Operating Conditions. J. Chromatogr., A 28, 1214, 81 89. 4. Decrop, W.; Gendeh, G.; Swart, R. Development of an Automated Method for Monoclonal Antibodies Purification and Analysis. Chromatography Today, Jun 29, 8 1. 5. Farnan, D.; Moreno, G.T. Multiproduct High-Resolution Monoclonal Antibody Charge Variant Separations by ph Gradient Ion-Exchange Chromatography. Anal. Chem. 29, 81(21), 8846 8857. 6. Farnan, D.; Moreno, D.T.; Stults, J.; Becker, A.; Tremintin, G.; van Gils, M. Interlaced Size Exclusion Liquid Chromatography of Monoclonal Antibodies. J. Chromatogr., A 29, 1216(51), 894 9. 7. Rea, J.C.; Moreno, G.T.; Lou, Y.; Parikh, R.; Farnan, D. High-Throughput Multi-Product Liquid Chromatography for Characterization of Monoclonal Antibodies. BioPharm International 21, 23, 44 51. 8. Grey, C.; Edebrink, P.; Krook, M.; Jacobsson, S.P. Development of a High Performance Anion Exchange Chromatography Analysis for Mapping of Oligosaccharides. J. Chromatogr., B Anal. Technol. Biomed. Life Sci. 29, 877, 1827 1832. Download your free copy of the Dionex Protein Therapeutics Application Notebook at www.dionex.com/proteintherapeutics. Corporate Headquarters Dionex Corporation 1228 Titan Way P.O. Box 363 Sunnyvale, CA 9488-363 Tel: (48) 737-7 Fax: (48) 73-943 Worldwide Sales and Service North America U.S./Canada (847) 295 75 South America Brazil (55) 11 3731 514 Europe Austria (43) 1 616 51 25 Benelux (31) 2 683 9768 (32) 3 353 42 94 Denmark (45) 36 36 9 9 France (33) 1 39 3 1 1 Germany (49) 6126 991 Ireland (353) 1 644 64 Italy (39) 2 51 62 1267 Sweden (46) 8 473 338 Switzerland (41) 62 25 9966 United Kingdom (44) 1276 691722 Asia Pacific Australia (61) 2 942 5233 China (852) 2428 3282 India (91) 22 2764 2735 Japan (81) 6 6885 1213 Korea (82) 2 2653 258 Singapore (65) 6289 119 Taiwan (886) 2 8751 6655 www.dionex.com Solutions The Dionex MAb Solutions enewsletter is your resource to discover new methods, applications, strategies and products to improve MAb analytics quality, throughput, and productivity. Subscribe to the free MAb Solutions enewsletter at www.dionex.com/mab. Dionex products are designed, developed, and manufactured under an ISO 91 Quality System. 211 Dionex Corporation CarboPac, Chromeleon, ProPac, and UltiMate are registered trademarks, and Amino Trap and MAbPac are trademarks of Dionex Corporation. PEEK is a trademark of Victrex PLC. LPN 271 5M 1/11 Printed in U.S.A. Passion. Power. Productivity.