of peptide mapping chemistries, 130Å or 300Å with tunable UV, photodiode array, or mass spectrometry

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1 UPLC PEPTIDE ANALYSIS SOLUTION UPLC Peptide Analysis Solution: n ACQUITY UPLC System n Peptide Separation Technology chemistries, 130Å or 300Å n Flexible detection capabilities, with tunable UV, photodiode array, or mass spectrometry n Robust software options for processing and managing data Conquering t he chromatographic challenges of peptide mapping Peptide mapping is an important technique for the comprehensive characterization of biopharmaceutical products. In a peptide map, each peptide fragment must be resolved into a single peak representing a significant chromatographic challenge due to the inherent complexity of protein digests. In addition to the large number of peptide fragments that are generated from the enzymatic digestion of a protein, the number of alternative peptide structures (posttranslational modifications, oxidations, etc.) can be equally daunting. Achieving adequate resolution in peptide maps is a tedious process. Good methods often have long run times and may not be as rugged as desired. Now you can easily obtain the resolution your lab needs. The Waters UPLC Peptide Analysis Solution, an Assured Performance Solution (APS) enabled by UltraPerformance LC technology, seamlessly combines analytical instrumentation with chemistry. This optimized and comprehensive system solution delivers the clear information you need so you will know as much as possible about your proteins.

2 Assured Performance Solutions Waters Assured Performance Solutions (APS) are a family of systems that bring together industry-leading technologies instrumentation, chemistries, software, qualification tools, and services that are specifically optimized for exceptional, application-focused performance. Designed by scientists for scientists, these solutions remove the guesswork in choosing the best solution for your application s needs. Expanding the boundaries resolution, reproducibility, and sensitivity The UPLC Peptide Analysis Solution brings a new level of resolution and sensitivity to peptide analysis that allows for more complete characterization of proteins. Significantly higher resolution UPLC delivers remarkable improvements in peptide mapping compared against HPLC. Exceptional resolution is possible because separations obtained through the use of UPLC s small particles result in dramatically narrower peaks. Just as significant, the surface chemistry of our Peptide Separation Technology packing material provides exceptional peak shape for a wide range of peptide structures. This remarkable column performance can only be achieved with the optimized instrumentation of the ACQUITY UPLC System, which features reduced system volumes, minimal detector band-broadening and accelerated data acquisition rates required to preserve high-efficiency separations while maximizing sensitivity. Whether you work in pharmaceuticals, proteomics, chemical analysis, or beyond, with Waters APS, you can be assured that whenever a new application challenge arises, Waters will have the technology and expertise to help you conquer it. Improved resolving power in peptide maps 1.2e-1 HPLC e-2 UPLC 7.0e More small peaks are resolved when the tryptic digest of Alcohol Dehydrogenase is separated with UPLC. Intelligently designed for optimum performance Waters scientists considered every aspect of peptide analysis from chemistries, instrumentation, software and methodology to usability, documentation, application support, and remote services to create this complete application solution. The UPLC Peptide Analysis Solution, based on our award-winning ACQUITY UPLC System separation technology and new Peptide Separation Technology Columns, provides the ultimate in high-resolution chromatographic performance. And the system offers flexible detection options such as Tunable UV (TUV), Photodiode Array (PDA), and Mass Spectrometry (MS) that are all optimized for UPLC.

3 Reproducibility Robust quantitation of trace peptides Advanced fluidic control of flow rate and gradient formulation combines with stable column chemistry to provide reproducible maps. Long-term assay stability is ensured with columns that are quality-control tested using a peptide map. 2.0e-1 1.5e Qualitative and quantitative analysis of variant peptides All elements of the UPLC Peptide Analysis Solution contribute to the successful qualitative and quantitative analysis of trace peptides. With this combination, variant proteins can be detected and measured with the greatest of confidence. n UPLC-based fluidic performance ensures retention time reproducibility. n High-resolution UPLC separations of both large and minor peaks enable accurate integration. n Peptide Separation Technology particles deliver chromatographic resolution and undistorted peak shape at extreme molar ratios. n The system s advanced optical and mass detection technologies have a wide dynamic range ensuring that peptides, at these extreme molar ratios, are detected even at the lowest limits of quantitation e-1 1.8e-1 1.7e-1 1.6e-1 1.5e-1 1.4e-1 1.3e-1 1.2e-1 1.1e-1 9.0e-2 7.0e The protein digest, spiked with a trace peptide, was separated with a Peptide Separation Technology Column. A small trace peptide can be identified and quantitated as shown here down to a 0.2 level. To achieve this quantitation, sensitivity, and resolution, peak shape must be maintained over a wide mass load. * * Do more with your results Waters software enables you to capitalize on your UPLC results. The Waters Laboratory Informatics Suite includes proven information management solutions that integrate all your lab resources in a consistent, scalable, and seamless application environment. Empower Software provides control of the ACQUITY UPLC System, including data acquisition, processing, and reporting, all in the market-leading chromatography data software (CDS) platform. Empower Software is scalable from a single workstation to an enterprise-wide CDS solution, and offers advanced features for method development, reporting, and custom calculations, as well as Enterprise Options for data management and automated method validation management. MassLynx Software, for advanced mass spectrometry applications, enables researchers to acquire, analyze, manage, and distribute information. MassLynx Software offers flexible control over complex instrument configurations, and Application Managers that are tailored for specific MS analyses. Whether your peptide analysis configuration requires CDS software for LC-based analysis, or is MS-based and can take advantage of Application Managers such as BiopharmaLynx for customized UPLC/MS biopharmaceutical data processing, you will be better able to manage that information with NuGenesis SDMS. SDMS is a scientific information management platform that automatically captures and stores data. SDMS makes information easier to access, facilitates regulatory compliance, and aids in the administration of intellectual property. SDMS converts scientific results into accessible and valuable corporate knowledge.

4 APPLYING UPLC TO YOUR PEPTIDE MAPS The UPLC Peptide Analysis Solution can be used the way you want to work. Flexibility in improving peptide mapping Peptide mapping is an essential technique for characterizing proteins. While most labs want to improve their assays, each has different criteria for what improved peptide mapping means. Whether you require more resolution, faster throughput, or need to focus on a particular aspect of a protein, the UPLC Peptide Analysis Solution can be used, without modification, to improve the peptide maps in the manner in which you choose. 5.5e-2 4.5e-2 3.5e-2 2.5e-2 1.5e-2 5.0e-3 HPLC Conventional peptide map Separation of a trypic digest of Phosphoylase b by HPLC. 7.0e-2 UPLC More resolution in same time Separation of a trypic digest of Phosphoylase b by UPLC with same gradient and same analysis time useful when more resolution is required without an increase in analysis time e-2 7.0e-2 UPLC Similar resolution in less time Separation of a trypic digest of Phosphoylase b by UPLC with a shorter column and scaled gradient provides the ability to reduce run times while maintaining resolution

5 Focused assay Modified peptides need to be resolved from native peptides for quantitiation to be acheived. A focused assay utilizing UPLC allows the run times to be reduced without any loss in resolution or quantitative information of the modified peptides. 9.0e-3 8.0e-3 7.0e nm 6.0e-3 5.0e T54, Oxidized m/z T m/z T m/z Focused assay at 12.5 to 16 B in 7.67 min, 50 mm, at 34 C, total run time min. Enhanced MS detection UPLC is an ideal inlet to MS. Separation based on small particles yields MS spectra that are more easily interpreted because the components are better resolved. Small particles result in small peak volumes which provide an increase in signalto-noise. It is easier, therefore, to detect and identify trace components of your peptide digest. Waters Peptide Separation Technology Columns are an integral part of the UPLC Peptide Analysis Solution. Their unique surface chemistry delivers exceptional peak shape for peptides over a wide range of molecular properties.

6 Improved glycopeptide mapping The analysis of site-specific glycosylation is challenging because glycopeptides often elute as broad, overlapping peaks. The surface chemistry of Peptide Separation Technology Columns yields sharper and more symmetrical peaks, simplifying this analysis. When this chromatographic separation is interfaced with a mass spectrometer, structurally informative fingerprints can now be routinely obtained. Successful separation of glycoforms Choices for method development with Peptide Separation Technology chemistry The columns are available with either 130 or 300Å pore sizes and are optimized for either trifluoroacetic acid (TFA) or formic acid (FA) modified mobile phases. All Peptide Separation Technology Columns are quality control tested and certified using a peptide digest to ensure reproducibility. Two pore sizes for flexible method development 1.9e-2 1.8e-2 1.7e-2 1.6e-2 1.5e-2 IgG Glycopeptide UPLC UV G1A G1B G0 1.2e-1 Fragment Size MW A 4 residues B 25 residues C 20 residues A B ACQUITY UPLC BEH 130Å C 1.4e-2 1.3e-2 1.2e-2 1.1e-2 9.0e-3 G IgG Glycopeptide UPLC/MS A B C ACQUITY UPLC BEH 300Å G2 (M+H) G1 (M+H) Comparison of the separation of tryptic digest of Phosphorylase b on ACQUITY UPLC BEH130 and BEH µm Peptide Separation Technology Columns G0 (M+H) A tryptic digest of mouse IgG was separated using a Peptide Separation Technology Column. The glycoforms were observed in both the UV and MS traces. The extracted ion chromatogram provides identification of the glycoforms and show that the two positional isomers of the G1 were separated. We use the Waters ACQUITY UPLC System for determining the primary sequences of antibodies as well as glycosylation and other secondary modifications. The UPLC separation provides sharp peaks, even when the quantities available are small. Dingyi Wen, Ph.D., Principle scientist, Biogen Idec Dr. Wen investigates antibody candidates by digesting them with endoproteases, followed by separation, and finally identification of peptides in the mass spectrometer. HPLC does not provide the resolution or sensitivity needed for the research protocols, and its use of splitting can result in loss of material, she explained.

7 Good science means good business To achieve your goals, your laboratories must operate at their best. You are balancing short-term financial performance against long-term business goals. You need to meet ever-aggressive product availability and delivery timetables, which require shorter turnaround times from product development through production. The Waters UPLC Peptide Analysis Solution simultaneously addresses laboratory productivity, efficiency, and analytical quality. Accelerate protein characterization An inherent degree of structural heterogeneity occurs in proteins. You need to isolate and identify all the protein s variants and degradation products. Utilizing peptide chromatography to describe protein heterogeneity patterns is a critical tool supporting effective discovery and lead development efforts. Waters technologies help you characterize proteins more comprehensively and efficiently, potentially accelerating your submissions to regulatory agencies. Manage risk Are you concerned by the demands to document product quality throughout the development process? The requirements are stringent, and remain applicable throughout the product s life cycle. Waters combines instruments, chemistries and software into performance assured solutions that you can rely on to give you consistent results for your validated analytical protocols. And because your scientists know more about their proteins, you can be more confident of your product and avoid costly surprises during regulatory review. Confirm quality Your objective is to demonstrate process consistency. Yet you must manage the fact that the product is heterogeneous, so your analytical methods are often complex. Concurrently, you have a production schedule to meet, so any delays in your part of the process are expensive. The UPLC Peptide Analysis Solution provides the flexibility you need to implement effective methods. You can rely on Waters to build in the product consistency you need for validated protocols. And when your analyses are complete, you can utilize Waters informatics solutions to archive the results for easy future access. Avoid costly retesting What is the impact of a failed analysis? The cost of a failed manufacturing process could be substantial. It may result in improperly discarding an entire process run. Or worse, it could lead to unnecessary checks to ensure that the manufacturing process has not developed issues. You need analytical methods that are robust and reliable. By testing our separation chemistries on ACQUITY UPLC Systems, following the same protocols Waters engineers used to qualify your system, Waters assures the reproducibility and ruggedness of your system. You will obtain the best information about your product, enabling you to support lot release requirements.

8 Sales Offices: Austria Australia Belgium and Luxembourg Brazil Canada China Czech Republic Denmark Finland France Germany Hong Kong Hungary India Ireland Italy Japan Korea Mexico The Netherlands Norway Poland Puerto Rico Russia/CIS / Singapore Spain Sweden Switzerland Taiwan UK US Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: Waters, ACQUITY, ACQUITY UPLC, UltraPerformance LC, UPLC, and NuGenesis are registered trademarks of Waters Corporation. BiopharmaLynx, Empower, MassLynx, and The Science of What s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation. Printed in the U.S.A. January EN LB-CP