Agilent BioHPLC Columns PROTEIN IDENTIFICATION AND IMPURITY PROFILING USING REVERSED-PHASE HPLC/UHPLC

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1 Agilent BioHPLC Columns PROTEIN IDENTIFICATION AND IMPURITY PROFILING USING REVERSED-PHASE HPLC/UHPLC

2 REVERSED-PHASE HPLC/UHPLC Agilent can boost your accuracy and productivity Reversed-phase is used to confirm protein identity, impurity profiling, and quantify post-translational modifications. The technique separates on the basis of differences in hydrophobicity, and uses denaturing conditions. This provides information about the molecule s primary ao acid sequence, as well as variations and modifications to the sequence. Agilent offers the most comprehensive range of wide-pore, 3Å, Å, and larger, reversed-phase BioHPLC columns, all backed by technical support experts and application chemists around the globe. The family includes.8, 3., and µm totally porous particles for pressures from to bar, Poroshell (superficially porous) particles for UHPLC separations at lower pressure, and polymeric columns for analysis under the most extreme conditions.

3 Agilent AdvanceBio RP-mAb columns: based on Poroshell technology with unique engineering for pore size and bonded phase, these columns deliver higher resolution and faster run times to provide accurate, reproducible results when analyzing intact monoclonal antibodies and mab fragments. Agilent AdvanceBio Peptide Mapping columns: quickly resolve and identify ao acid modifications in primary structure. With their.7 μm particles and C8 functionality, AdvanceBio Peptide Mapping columns deliver excellent retention, resolution, and peak shape for basic hydrophobic peptides. Agilent Poroshell 3 columns: the industry s first superficially porous small particle columns for fast polypeptide and protein separations. Agilent ZORBAX RRHD 3Å.8 µm columns: deliver UHPLC performance for reversed-phase separations of intact proteins, protein fragments, and digests with bar stability. Agilent ZORBAX 3Å 3. & µm columns: fully porous materials for HPLC and prep separations; many of the bonded phases are scalable from the.8 µm particle. Agilent PLRP-S columns: macroporous polymer particles deliver HPLC separations over the widest ph range. With 3 wide-pore sizes and 8 particle sizes, the PLRP-S columns provide optimum solutions for analytical prep separations of peptides, proteins, and protein complexes. INSIDE AdvanceBio RP-mAb Columns For intact monoclonal antibodies and mab fragments Page AdvanceBio Peptide Mapping Columns For basic hydrophobic peptides Page 7 Poroshell 3 Columns For intact protein separations Page ZORBAX RRHD Columns For intact proteins and peptide digests Page ZORBAX 3StableBond Columns For low-ph separations Page 7 PLRP-S Columns For stability under extreme conditions Page 8 Column Selection Guide/ Ordering Information Page To learn more about perforg high-resolution protein separations with Agilent reversed-phase columns, visit agilent.com/chem/advancebio 3

4 REVERSED-PHASE HPLC Which Fast LC column is best for your reversed-phase separation? Agilent offers the widest range of fast HPLC/UHPLC wide-pore columns. So you have the flexibility to create methods with maximum resolution, whether you have a, 6, or bar instrument. Wide-pore, 3Å columns are necessary for efficient separation of proteins and peptides because they allow these analytes to completely access the bonded phase. Reversed-Phase Column Selection Application Agilent Columns Notes Intact monoclonal antibodies and mab fragments Intact proteins, monoclonal antibodies, mab fragments and polypeptides Large intact proteins, monoclonal antibodies Peptides AdvanceBio RP-mAb, Å, 3. μm SB-C8 C Diphenyl Particles with a wide pore diameter are necessary for an efficient separation of large biomolecules such as intact mabs, as they allow the analytes to completely access the bonded phase. The use of Poroshell technology, with reduced diffusion distances, enhances this efficiency even further. C chemistry is well suited to mab separations, providing stability at low ph and compatibility with methods that require USP L6 columns. StableBond C8 gives scalability and method transfer. The Diphenyl phase, unique to Agilent, offers alternative selectivity. ZORBAX 3Å,.8 μm Optimized packing processes achieve stability up to, bar for use with the Agilent 9 Infinity LC. RRHD.8 μm columns are available in and mm lengths for fast or high RRHD 3SB-C8 resolution truly high definition separations of the most complex samples. StableBond C8 is RRHD 3SB-C8 ideal for complex protein and protein digest separations. RRHD 3SB-C3 RRHD 3-Diphenyl ZORBAX 3Å, 3. and µm 3SB-C8 3SB-C8 3SB-C3 3SB-CN ZORBAX 3Å, Extend-C8 Poroshell 3 3SB-C8 3SB-C8 3SB-C3 3Extend-C8 AdvanceBio Peptide Mapping Ideal for use with HPLC systems. StableBond C3 and CN are useful for larger, more hydrophobic compounds. Incorporate a unique bidentate silane, combined with a double-endcapping process that protects the silica from dissolution at high ph up to ph.. Poroshell columns use a unique particle made with a layer of porous silica on a solid core of silica. This reduces the diffusion distance for proteins, making possible practical, rapid HPLC separations of peptides and proteins. An ideal Å pore size for identifying a wide molecular weight range of peptides. Tested with a challenging peptide mix to ensure performance. The unique Agilent Poroshell technology enables higher flow rates and better resolution of the full peptide sequence. Peptides to DNA PLRP-S Particles are inherently hydrophobic and so an alkyl ligand bonded phase is not required for reversed-phase separations. This gives a highly reproducible material Å that is free from Å silanols and heavy metal ions. 3Å Å Å Small molecules/synthesis PLRP-S Å Recombinant peptides/ PLRP-S 3Å proteins Large proteins PLRP-S Å DNA/high speed separation PLRP-S Å Ao acids ZORBAX Ao Acid Analysis (AAA) Tested for ao acid analysis using well-known OPA and FMOC precolumn derivatization chemistry. Options for HPLC and UHPLC available.

5 ADVANCEBIO RP-mAb COLUMNS RESOLVE mabs FASTER AND BETTER The Agilent AdvanceBio RP-mAb column delivers higher resolution and faster run times to provide accurate, reproducible results when analyzing monoclonal antibodies for biopharma discovery, development, and QA/QC applications. Exclusive Agilent Poroshell technology, built into every AdvanceBio RP-mAb column, gives you the advantages of: Improved accuracy: Superficially porous particles (3. μm) with wide pores (Å) increase mab resolution while maintaining compatibility with all LC instruments Speed: Shorter analysis times compared to columns packed with fully porous particles of the same size (Figure ) Lower costs: The robust Poroshell packed bed and μm inlet frit extend column lifetime by helping prevent inlet blockage Flexible method development: Range of chemistries SB-C8, C, and diphenyl Sharp peaks with fine detail for short runs Characterization in less than utes AdvanceBio RP-mAb C Method Parameters Column dimensions:. x mm, 3. µm Mobile phase A:.% TFA in water:ipa (98:) Mobile phase B: IPA:ACN:Mobile phase A (7::) Flow rate:. ml/ -8% B in, wash at 9% B, re-equilibration at % B μl injection of humanized recombinant Herceptin Variant IgG intact from Creative Biolabs ( mg/ml) Temperature: 8 C Detection: nm Figure. Here, an AdvanceBio RP-mAb C column delivered excellent peak shape and detailed resolution of intact humanized recombinant Herceptin IgG in less than utes. Agilent AdvanceBio vs. the competition Superior to other protein columns AdvanceBio RP-mAb C Other manufacturer's column C, Å, 3. µm Other manufacturer's column C, Å, 3.6 µm Other manufacturer's column C, 3Å,.6 µm Method Parameters Column dimensions:. x mm, 3. µm Mobile phase A:.% TFA in water:ipa (98:) Mobile phase B: IPA:ACN:Mobile phase A (7::) Flow rate:. ml/ -8% B in, wash at 9% B, re-equilibration at % B μl injection of humanized recombinant Herceptin Variant IgG intact from Creative Biolabs ( mg/ml) Temperature: 8 C Detection: nm Figure. Specifically designed for mab separatioins, AdvanceBio RP-mAb provides superior peak shape and resolution than other columns used for intact protein separations. To learn more about perforg high-resolution protein separations with Agilent reversed-phase columns, visit agilent.com/chem/advancebio

6 Exceptional speed and confidence for mab separations As with all columns manufactured by Agilent, AdvanceBio RP-mAb columns undergo rigorous end-to-end QC testing to ensure reproducibility and performance. In this example (Figure 3), intact humanized recombinant Herceptin IgG was characterized using an AdvanceBio RP-mAb Diphenyl column. The unique diphenyl phase resolves even more fine detail. Figure demonstrates how the wide-pore Poroshell technology of the AdvanceBio RP-mAb column delivers high efficiency, a short analysis time, and low pressure, at temperatures below 8 C the typical temperature of many reversed-phase methods. Selective diphenyl phase More fine details resolved AdvanceBio RP-mAb Diphenyl Method Parameters Column dimensions:. x mm, 3. µm Mobile phase A:.% TFA in water:ipa (98:) Mobile phase B: IPA:ACN:Mobile phase A (7::) Flow rate:. ml/ -8% B in, wash at 9% B, re-equilibration at % B μl injection of humanized recombinant Herceptin Variant IgG intact from Creative Biolabs ( mg/ml) Temperature: 8 C Detection: nm Figure 3. The unique selectivity of AdvanceBio RP-mAb Diphenyl resolves even more fine detail. The Poroshell advantage High accuracy, low backpressure 3 3 AdvanceBio RP-mAb SB-C8 Pressure: bar Method Parameters Column dimensions:. x mm, 3. µm Mobile phase A:.% TFA in water Mobile phase B: n-propanol:acn:mobile phase A (8::) Flow rate:.8 ml/ -% B in, wash at 9% B, re-equilibration at % B μl injection of Fc/Fab, papain-digested humanized recombinant Herceptin Variant IgG from Creative Biolabs ( mg/ml) Temperature: 6 C Detection: nm Figure. AdvanceBio RP-mAb columns perform well at temperatures below 8 C. 6

7 ADVANCEBIO PEPTIDE MAPPING COLUMNS Reduce peptide mapping time without losing resolution Agilent AdvanceBio Peptide Mapping columns let you quickly resolve and identify ao acid modifications in primary structure, unlike fully porous columns which can take 6 utes. These advanced biocolumns feature a Å pore size with superficially porous.7 μm particles, and are designed to deliver: Greater analytical confidence: Each batch of AdvanceBio Peptide Mapping media is tested with a rigorous peptide mix to ensure suitability and reproducibility, and to enable the identification of key peptides in complex peptide maps. Time savings: Perform high-resolution separations to 3 times faster than with fully porous HPLC columns. Every instrument works harder:.6, 3., and. mm id columns are stable to 6 bar, enabling you to get the most from your UHPLC instruments. They can also deliver excellent performance for your legacy bar instruments, too. More flexibility: Increase MS sensitivity with formic acid mobile phases on any HPLC. Quality assurance testing with peptide mix Column: AdvanceBio Peptide Mapping,. x mm,.7 µm, p/n Flow rate:. ml/ Injection: µl Temp: ºC Pk no. MW Component 77 Bradykinin fragment -7 6 Bradykinin 3 6 Angiotensin II 673 Neurotensin 9 Angiotensin I 6 6 ACTH fragment Renin 8 8 Melittin 7 Detection: nm A, water (.% TFA), B, ACN (.8% TFA), -,- 6% B; -6, 6-9% B Agilent Peptide Mapping Standards Mix (.-. μg/μl per peptide) p/n 9-83 Figure. Test mix used for every batch of AdvanceBio Peptide Mapping media. The mixture contains 8 hydrophilic, hydrophobic, and basic peptides, ranging in molecular weight from 77 Da to 8 Da. Every column is also tested with a smallmolecule probe to ensure efficiency. 8 PEPTIDE MAPPING STANDARDS Now Available p/n 9-83 Peptide map of a biosimilar EPO Column: Flow rate: Injection: 3 AdvanceBio Peptide Mapping,. x mm,.7 µm, p/n ml/ µl (. mg/ml) Temp: ºC Detection: nm A, w n, 6-9% B 3 Figure 6. AdvanceBio Peptide Mapping column easily confirms protein identity and identifies all post-translational modifications. x 6 Cpd :.: +ESI ECC Scan Frag=7.V EPOdigestd.d Cpd : Counts vs. Acquisition Time () Figure 6a. EPO digest, LC/MS TOF 9% sequence coverage achieved using MassHunter Workstation software. To learn more about perforg high-resolution protein separations with Agilent reversed-phase columns, visit agilent.com/chem/advancebio 7

8 Excellent reproducibility The science behind AdvanceBio columns helps increase accuracy and productivity to support faster biopharmaceutical analysis and efficiency. In addition, AdvanceBio columns are rigorously tested by Agilent to ensure reproducibility, giving you greater confidence in your results. Figure 7 demonstrates the superior lot-to-lot and run-to-run reproducibility that can be achieved using AdvanceBio Peptide Mapping columns. Lot-to-lot reproducibility after injections Norm Silica lot PEP79 Inj. (39 bar) Inj. (388 bar) 3 Injection RT () RT3 () RT () RT () Injection PW PW3 PW PW Norm Inj. (397 bar) Norm Silica lot B69 3 Inj. (397 bar) Inj. ( bar) Injection RT () RT3 () RT () RT () Injection PW PW3 PW PW Column: AdvanceBio Peptide Mapping,. x mm,.7 µm, p/n 67-9 Flow rate:. ml/. Injection: µl Temp: C Detection: nm A, water (.% TFA), B, ACN (.8% TFA), -8, -6% B; 8.-9, hold 9% B Sigma HPLC peptide standards: -Gly-Tyr, -Val-Tyr-Val, 3-Met Enk, - Angio II, - Leu Enk Figure 7. A. x mm AdvanceBio Peptide Mapping column was used for maximum resolution. 8

9 Ideal for fast or high resolution peptide separation Agilent AdvanceBio Peptide Mapping columns are made with.7 μm ultra-high purity (>99.99% SiO ) superficially porous silica, and are densely bonded with C8 to provide the high selectivity needed for peptide separations. This type of particle delivers high efficiency at lower pressures when compared to small, totally porous particles. In Figure 8, you can see how AdvanceBio Peptide Mapping columns ensure reproducibility of peak heights and retention times for more accurate target peptide identification. LC/MS reproducibility x Column: AdvanceBio Peptide Mapping, 3. x mm,.7 µm, p/n LC/MS (Agilent 6 Q-TOF) Parameters Dry gas: L/, Vcap: V, Fragmentor: V Flow rate:.3 ml/ Injection: µl Temp: C A, water (.% FA), B, ACN (.% FA), -3, % B; 3-3, -% B; 3-, -6% B;.-7., hold 9% B Stratagene mab, in-house tryptic digestion Figure 8. This entire IgG tryptic peptide map was completed in just utes (n=). To learn more about perforg high-resolution protein separations with Agilent reversed-phase columns, visit agilent.com/chem/advancebio 9

10 POROSHELL 3 COLUMNS Quick, confident separation of intact proteins and protein fragments Agilent Poroshell columns are the ideal choice for separating and characterizing complex bio-molecules, including intact and protein fragments at pressures up to bar. For fast analysis of intact proteins, we recommend Agilent Poroshell 3 columns. The Poroshell 3 superficially porous particle is a revolutionary chromatography material that produces very fast, high-resolution, RP-HPLC separations of proteins and other macromolecules. Poroshell columns work so well for fast separations of macromolecules because of their rapid mass transfer into and out of their thin 3Å porous shell, providing sharper peaks for higher resolution, for improved accuracy of impurity profiling and post-translational modifications. Shorter analysis time, higher resolution, with lower column pressures Feature. µm, 3Å porous shell on solid particle Advantage Shorter diffusion distances for reduced analysis times μm particle Lower operating pressure Long column lifetime due to reduced sample trapping UHPLC resolution and efficiency at lower pressures, for faster separations. µm. µm Poroshell 3. µm StableBond chemistry Proven stability at low ph Long column lifetime with TFA and formic acid Use Poroshell 3 columns for analyzing intact proteins and large peptide fragments.

11 High flow rates with. mm id Poroshell 3 columns, with their larger 3Å pore size and thin shell, are a reliable choice for fast separations of intact proteins. The separation shown in Figure 9 was completed in less than. utes. With the rapid mass transfer capability of the superficially porous particle, Poroshell 3 columns are the best columns for high efficiency at higher flow rates for extremely rapid protein separations. Separation of peptides and proteins 3. Angiotensin II. Neurotensin 3. RNase. Insulin. Lysozyme 6. Myoglobin 7. Carbonic anhydrase 8. Ovalbu.. Time () Figure 9. Separation of 8 peptides and proteins in under. utes good peak capacity for rapidly separating complex samples Column: Poroshell 3SB-C8,. x 7 mm, µm Mobile phase: A:.% TFA B:.7% TFA in ACN Flow rate: 3. ml/ Temperature: 7 C Detection: UV nm to % B in. Pressure: bar Separation of monoclonal antibody heavy and light chains Heavy chains Light chains 8 IgG treated with: DTT 6 8 Glycosylated Non-Glycosylated 8 IgG treated with: DTT Peptide-N-glycosidase F 6 8 IgG treated with: 8 DTT Peptide-N-glycosidase F Carboxypeptidase-B 6 8 Figure. Chromatographic comparison of antiboday lgg after reduction and enzymatic cleavage. Time () % Solvent B.... Column: Poroshell 3SB-C8,. x 7 mm, µm Mobile phase: A: H O-ACN (9:) B: H O-ACN (:9) A and B contain.% TFA and 3 ml/l of PEG 3 Flow rate:. ml/ Temperature: 7 C Detection: UV nm To learn more about perforg high-resolution protein separations with Agilent reversed-phase columns, visit agilent.com/chem/advancebio

12 Ultra fast separation advantage µm Poroshell 3 columns can deliver compelling fast-separation advantages versus a competitor s superficially porous 3.7 µm, mm (low flow) column. In Figure, the Poroshell 3 column maintained critical resolution at ultra-fast separation speeds with ballistic gradients while maintaining pressure drops for HPLC less than bar. The Agilent Poroshell 3 column resolves the 8 proteins x faster than the alternative superficially porous column. Poroshell 3 vs. Competitor Competitor column * 7 8 * Column: Competitor column: C8,. x mm, 3.7 µm Protein Standard Mix (3 kda- 66 kda) Mobile phase: A: water 8(.% TFA) B: ACN (.8% TFA) 8 Temperature: C DAD: UV nm -9% B, 6,.3 ml/ * 7. Ribonuclease A. Lysozyme 3. Cytochrome c. Insulin 7. Transferrin 6. Myoglobin 7. B-amylase 8. Thyroglobulin Agilent column x faster * * Column: Poroshell 3SB-C8,. x 7 mm, µm * The Agilent Poroshell 3 column Mobile phase: A: water (.% TFA) B: ACN (.8% TFA) provides better peak shape for Temperature: C improved accuracy in the analysis DAD: UV nm of peak, transferrin. -9% B,,. ml/ 8 Figure. Ultra-fast separation advantage with Poroshell 3 SB-C8 columns versus a competitor.

13 Bonded phase choices offer more resolving power and improved recovery Poroshell 3 HPLC columns are available in four bonded phases 3SB-C8, C8, C3, and 3Extend-C8. Reducing the bonded phase chain reduces the hydrophobicity of the 3SB-C8 and 3SB-C3 bonded phases. For example, insulin and cytochrome c are baseline resolved on the Poroshell 3SB- C3 column, while these same analytes co-elute on the Poroshell 3SB-C8 column under the conditions described in Figure. For some complex samples, protein recovery can be an issue. Using the less hydrophobic Poroshell 3SB-C8 and C3 columns has been shown to improve recovery. Figure 3 shows the traces that result from injecting μl of fermentation broth on a Poroshell 3SB-C3 column, followed by the clean trace immediately after a blank injection of μl of water. Improved sample recovery and resolution of critical pairs of peaks improves the accuracy of protein analytics. 7 Poroshell 3SB-C8, Columns: Poroshell 3SB-C8,. x 7 mm, µm Poroshell 3SB-C3,. x 7 mm, µm Mobile phase: A:.% TFA/H O B:.7% TFA/ACN Flow rate:. ml/ Temperature: 7 C to % B in 3. Detection: UV nm Poroshell 3SB-C Figure. Poroshell 3SB-C3 resolves peaks and 6, insulin and cytochrome c, which co-elute with the more hydrophobic C8 phase Angiotensin II. Neurotensin 3. RNase A. Insulin B chain. Insulin 6. Cytochrome c 7. Lysozyme 8. Myoglobin 9. Carbonic anhydrase A () 3 3 µl fermentation broth Improved accuracy and precision of analysis. Column: Poroshell 3SB-C3,. x 7 mm, µm Mobile phase: A:.% TFA /H O B:.7% TFA/ACN Flow rate:. ml/ Temperature: C to 6% B in., to % B in. Water injection (blank) immediately follows an injection of µl of clarified fermentation broth Detection: UV nm µl water blank 6 8 RT () Figure 3. No carryover when using the Agilent Poroshell 3SB-C3 column. To learn more about perforg high-resolution protein separations with Agilent reversed-phase columns, visit agilent.com/chem/advancebio 3

14 Achieve unique selectivity from ph -. Figure shows that selectivity differences, coupled with the high resolving power of Poroshell 3 columns, can help you achieve very favorable improvements in your separation. Poroshell 3Extend-C8 columns combine bidentate silane with a double-endcapping process that protects the silica from dissolution at high ph, to provide longer column lifetime and improve baseline at higher ph. Figure shows fast separation of small proteins and polypeptides in less than one ute, using the most hydrophobic phase, C8. SB-C Extend C SB-C SB-C Column: Poroshell 3,. x 7 mm, µm Degraded insulin Mobile phase: A: water (.% TFA) B: ACN (.8% TFA) Flow rate:.7 ml/ Temperature: C % B hold.3, -6% B,.7. Figure. Changing the bonded phase improves resolution of the critical pair of peaks to improve accuracy of analysis Time () Figure. Fast separation of small proteins and polypeptides in less than one ute..3. gly-tyr. mg/ml. Val-tyr-val. mg/ml 3. Met-enkephalin. mg/ml. Leu-enkephalin. mg/ml. Angiotensin II. mg/ml RNase A mg/ml 7. Cytochrome c mg/ml 8. Holotransferrin mg/ml 9. Apomyoglobin mg/ml Column: Poroshell 3SB-C8,. x 7 mm, μm peptides/proteins,. μl Mixer bypassed with P/N G3-673; Loop-bypass program Mobile phase: A:.% TFA, HO B:.7% TFA, ACN Flow rate: 3 ml/. -% B in.33 Temperature: 7 C Detector: DAD /6 nm, ref = 3/ nm

15 ZORBAX RRHD COLUMNS 3Å.8 µm particles ensure stability at bar Wide-pore ZORBAX RRHD 3SB-C8, C8, C3, and 3-Diphenyl.8 μm columns deliver UHPLC performance for separations of intact proteins and peptide digests. Together with UHPLC instruments, such as the Agilent 9 Infinity LC, these versatile columns enable higher order characterization and shorter analysis times. The Diphenyl phase provides unique selectivity, and ZORBAX StableBond technology (C8, C8, and C3) gives you the advantages of: Low ph stability, which lets you confidently perform protein and peptide separations down to ph using trifluoroacetic acid (TFA) and formic acid eluents. Temperature stability, up to 8 C, allows you to run separations at higher temperatures without compromising column lifetime. So you can improve efficiency and reduce eluent viscosity. Reproducibility and recovery of monoclonal antibodies For larger proteins, including monoclonal antibodies, a shorter, less hydrophobic C8 functionality is used. This provides improved resolution and high recovery. Column: ZORBAX RRHD 3SB-C8,. x mm,.8 µm mab Mobile phase: A: H O:IPA (98:),.% TFA B: PA:ACN:H O (7::),.% TFA Flow rate:. ml/ Temperature: 8 C Detection: 9 Infinity LC at nm ma 3 st injection th injection Time () % B Increasing protein size and hydrophobicity ma... 3 C8 C8 C3 Diphenyl With four different ligand types; C8, C8, and C3 alkyl chains; and diphenyl for additional selectivity based on pi-pi aromatic ao acids, Agilent has the widest range of reversed-phase columns for peptide and protein UHPLC separations. 7 3 UV pre- and post- injection column blank run overlays pressure 3 Figure 6. This example demonstrates the reproducibility and lifetime of an Agilent ZORBAX RRHD 3SB-C8 column over injections, with no retention time shifts or peak shape abnormalities. The bottom chromatogram shows the pre- and post- injection blank runs and gradient pressure curves, proving that there was no ghosting or pressure increase after injections therefore, no column failure or sample losses which will improve accuracy of quantitation. To learn more about perforg high-resolution protein separations with Agilent reversed-phase columns, visit agilent.com/chem/advancebio

16 More speed, more resolution The unique diphenyl phase was previously available only on smaller pore Å Pursuit XRs and Å Pursuit columns. Now, by applying this proven bonding chemistry to ZORBAX 3Å.8 μm columns, this unique selectivity can be used for larger protein separations..8 µm RRHD columns can deliver fast separation advantages over competitive 3.7 µm columns (low-flow model), showing appreciable gains in analysis speed while maintaining comparable resolution. In Figure 8,.8 µm columns maintained (and surpassed) critical resolution at ultra-fast separation speeds with ballistic gradients a demonstrable UHPLC advantage. ZORBAX RRHD vs. Competitor column Competitor column: C8,. x mm 6 bar 3 Fast separation of reduced monoclonal antibody Light chain Heavy chain.93 3 Light chain Heavy chain 3 Figure 7. Comparison of fast separation of reduced monoclonal antibody using Agilent ZORBAX RRHD 3SB-C3 and 3-Diphenyl,. x mm,.8 µm better resolution of the two heavy chains is obtained with the diphenyl column Heavy chain Columns: ZORBAX RRHD 3SB-C3 and 3-Diphenyl,. x mm,.8 µm Reduced monoclonal antibody (IgG) (. mg/ml) Sample injection: µl Mobile phase: A:.% TFA in water B: 8% n-propyl alcohol, % ACN, 9.9% water and.% TFA -% B, -% B, -% B Flow rate:. ml/ Temperature: 7 C Detection: UV 8 C3 Diphenyl Heavy chain ZORBAX RRHD 3SB-C8,. x mm,.8 µm 39 bar Mobile phase: Flow rate: Temperature: C DAD: nm Degraded insulin A: water (.% TFA) B: ACN (.8% TFA) 3% B hold % B,.3 ml/ Figure 8. Degraded insulin separation. Agilent Rapid Resolution High Definition 3Å.8 µm columns achieve superior bandwidths and peak shapes as the competition (improved resolution of degradation products). 6

17 ZORBAX 3Å 3. AND μm COLUMNS Extraordinary chemical and temperature stability in the ph -6 range Agilent ZORBAX 3StableBond columns are ideal for the reproducible separation of proteins and peptides for two reasons: Wide pore, 3Å columns allow proteins, peptides, and other large molecules to completely access the bonded phase. ZORBAX 3StableBond columns have unmatched durability with the low-ph mobile phases (including TFA) that are typically used for protein and peptide separations. For LC/MS separations at low ph, ZORBAX 3StableBond columns can also be used with formic acid and acetic acid mobile phase modifiers. These columns are available in four different bonded phases, StableBond C8, C8, C3, and Extend-C8 for selectivity and optimized recovery of proteins and polypeptides. To further increase sample recovery and improve efficiency for difficult proteins, 3StableBond columns can be used up to 8-9 C. Short-chain ZORBAX 3SB-C3 is stable at low ph and high temperature for reproducible separations and longer column lifetime Column: ZORBAX 3SB-C3,.6 x mm, μm % k Phenyltheptane Remaining ZORBAX 3SB-C3 Competitor C Mobile phase: Gradients -% B in 8 A:.% TFA in Water B:.% TFA in Acetonitrile Isocratic Retention Test Conditions: -phenylheptane % A, % B Flow rate:. ml/ Temperature: 6 C,, 3,,, 6, 7, 8, Volume of Mobile Phase (ml) Figure 9. Typical mobile phases for protein and peptide separations combine a very low ph with TFA (or other acids) with high temperature to denature and solubilize proteins. Agilent StableBond columns have extremely long lifetimes under these conditions. To learn more about perforg high-resolution protein separations with Agilent reversed-phase columns, visit agilent.com/chem/advancebio 7

18 PLRP-S HPLC COLUMNS Reproducible separations under extreme conditions The Agilent PLRP-S column family includes a range of pore and particle sizes, all with identical chemistries and fundamental chromatographic characteristics. They feature: Durable, resilient polymer particles that deliver reproducible results for longer column lifetimes Thermal and chemical stability for separations at extremes of ph and high temperature -Å pore sizes that provide high efficiency separations over the full range of protein and peptide sizes PLRP-S particles are inherently hydrophobic; and so no bonded phase, alkyl ligand, is required. This ensures a highly reproducible material that is free from silanols and heavy metal ions. In addition, PLRP-S provides scalability from analytical separations through purification, prep columns, and bulk media. When purifying proteins it may be necessary to sanitize the column with the PLRP-S material. It is possible to use extremely aggressive cleaning in place, including M NaOH, as demonstrated in Figure. Media can be cleaned in a packed column (CIP), or in bulk, using a range of solubilizing agents such as sodium hydroxide to ensure unsurpassed column/media lifetimes. Large fibrous proteins Exploiting chemical stability NaOH concentration. Collagen (, MW). Fibrinogen (3, MW) Before After column volume of M NaOH 3 3. Oxytocin. Angiotensin II 3. Angiotensin I. Insulin PLRP-S 3Å 8 PLRP-S Å Column: Mobile phase: PLRP-S 3Å.6 x mm, - μm A:.% TFA in Water B:.% TFA in ACN Flow rate: Detector: -% B in s. ml/ UV nm Column: Column: PLRP-S 3Å.6 x mm, 8 μm PLRP-S Å.6 x mm, 8 μm Mobile phase: Flow rate: Detector: A:.% TFA in wate B:.% TFA in % water: 9% ACN. ml/ -6% B in UV, nm Figure. The Agilent PLRP-S media is chemically robust and can withstand extremely aggressive sanitizing/cleaning protocols, to ensure unsurpassed column/media lifetimes. Figure. Agilent PLRP-S 3Å and PLRP-S Å material separate large fibrous proteins, as shown here. However, improved peak shape and increased peak height were obtained from the larger pore size PLRP-S Å column. 8

19 INSTRUMENTS FOR PROTEIN IDENTIFICATION AND IMPURITY PROFILING Agilent 6 Infinity Bio-inert Quaternary LC System: your best choice for protein separations Agilent 9 Infinity Binary LC System: most adaptive UHPLC system with the widest application range Use for protein identification for best results use with Poroshell 3 The only UHPLC that provides a metal-free sample flow path. Other advantages include: % Bio-inertness No stainless steel: sample does not touch metal surfaces ph to ph 3 (ph short-term) Handles M salt and 8 M urea New capillary technology UHPLC capability: 6 bar Robust and easy to use with low surface activity, corrosion resistance, active seal wash, and quaternary buffer mixing Use for impurity profiling, peptide mapping or ultrafast gradients for best results use with ZORBAX RRHD 3Å.8 µm Best-in-class performance in terms of resolution per time, dispersion, sensitivity, accuracy and precision in LC/UV and LC/MS. By combining innovative active damping, microfluidic mixing and optofluidic waveguides detection technology: UHPLC power range with up to bar and ml/ The fastest and easiest method transfer using ISET, Agilent s unique Intelligent System Emulation Technology UHPLC productivity, HPLC-like costs of ownership Agilent 6 Infinity Binary LC System: raising the standard in analytical HPLC 6 bar,high-speed 8 Hz detector, up to x higher sensitivity % HPLC compatibility, UHPLC capability: UHPLC performance, HPLC-like costs of ownership Agilent 9 Infinity Quaternary LC System: combine performance with flexibility The only quaternary UHPLC system with binary-like accuracy and precision. Further advantages include: UHPLC power range with up to bar and ml/ BlendAssist, the easiest tool for accurate buffer and additive blending Use for any kind of standard UHPLC application Support of LC and LC/MS applications, with any narrow and standard bore analytical column (. -.6 mm id) Superior gradient accuracy by high-pressure mixing Use for method development or walk-up systems with accurate buffer blending UHPLC productivity, HPLC-like costs of ownership To learn more about perforg high-resolution protein separations with Agilent reversed-phase columns, visit agilent.com/chem/advancebio 9

20 Ordering Information and Specifications AdvanceBio RP-mAb columns Bonded phase Pore size Temp. limits ph range Endcapped C Å 9 C. to 8. Yes SB-C8 Å 9 C. to 8. No Diphenyl Å 9 C. to 8. Yes Description Size (mm) Particle Size (μm) Part Number C. x C. x C. x C. x C.6 x C.6 x C.6 x SB-C8. x SB-C8. x SB-C8. x SB-C8. x SB-C8.6 x SB-C8.6 x SB-C8.6 x Diphenyl. x Diphenyl. x Diphenyl. x Diphenyl. x Diphenyl.6 x Diphenyl.6 x Diphenyl.6 x Poroshell 3 columns for protein analysis Bonded phase Pore size Temp. limits ph range Endcapped 3SB-C8, C8, C3 3Å 9 C. to 8. No 3Extend-C8 3Å C above ph 8, 6 C below ph 8. to. Yes Description Size (mm) 3SB-C8 USP L 3SB-C8 USP L7 Capillary. x Capillary. x SB-C3 3Extend-C8 USP L MicroBore. x Narrow Bore. x Guard cartridge, /pk. x Guard hardware kit MicroBore guard, 3/pk. x

21 ZORBAX 3Å columns for HPLC and UHPLC protein separations Bonded phase Pore size Temp. limits ph range Endcapped 3SB-C8 3Å 9 C. to 8. No 3SB-C8 3Å 8 C. to 8. No 3SB-C3 3Å 8 C. to 8. No 3SB-CN 3Å 8 C. to 8. No 3Extend-C8 3Å 6 C. to. Double 3-Diphenyl 3Å 8 C. to 8. Yes Description Size (mm) Particle Size (μm) 3SB-C8 USP L 3SB-C8 USP L7 3SB-CN USP L 3SB-C3 USP L6 3Extend-C8 USP L 3-Diphenyl USP L MicroBore. x MicroBore RR. x MicroBore RR. x Narrow Bore. x Narrow Bore. x Narrow Bore. x Narrow Bore. x Narrow Bore RR. x Narrow Bore RR. x Narrow Bore RR. x Solvent Saver Plus 3. x Solvent Saver Plus 3. x Analytical.6 x Analytical.6 x Analytical.6 x Rapid Resolution.6 x Rapid Resolution.6 x Rapid Resolution.6 x Semi-Preparative 9. x MicroBore Guard, 3/pk. x Guard Cartridge, /pk.6 x Guard Cartridge, /pk. x PrepHT Cartridge. x PrepHT Cartridge. x PrepHT Cartridge. x PrepHT Cartridge. x PrepHT Cartridge. x PrepHT Endfittings, /pk PrepHT Guard Cartridge, /pk 7 x Guard Cartridge Hardware To learn more about perforg high-resolution protein separations with Agilent reversed-phase columns, visit agilent.com/chem/advancebio

22 PLRP-S HPLC columns for widest ph range Description Size (mm) Particle Size (μm) PLRP-S Å USP L PLRP-S 3Å USP L MicroBore. x 3 PL3-3 PL3-3 PLRP-S Å USP L MicroBore. x PL3- PL3- MicroBore. x 3 PL3-33 PLRP-S Å USP L Analytical.6 x 8 PL-8 PL-8 PL-83 Analytical.6 x PL- PL- Analytical.6 x PL-3 PL-3 Analytical.6 x PL- PL- PL- PL-3 Analytical.6 x 3 PL-33 PL-33 Analytical.6 x 3 PL-3 PL-3 Analytical. x 8 PL9-8 Analytical. x 8 PL9-38 PL9-38 PL9-383 Analytical. x 8 PL9-8 PL9-8 PL9-83 Analytical. x PL9- PL9- Analytical. x PL9-3 PL9-3 Analytical. x PL9- PL9- PL9- PL9-3 Analytical. x 3 PL9-33 PL9-33 Analytical. x 3 PL9-3 PL9-3 Method development.6 x 3 PL-7 PL Method development.6 x - PL- PL- Method development.6 x - PL- PL- Method development.6 x PL- PL- PL- PL-3 Method development.6 x 8 PL-8 PL-8 PL-8 Method development.6 x 3 PL-37 PL-373 Method development.6 x - PL-3 PL-3 Method development.6 x - PL-3 Method development.6 x PL-3 PL-3 PL-3 PL-33 Method development.6 x 8 PL-38 PL-38 PL-38 PL-383 Prep to Process x 3 3 PL8-3 PL8-33 Prep to Process x 3 - PL8-6 PL Prep to Process x 3 - PL8-6 PL Prep to Process x 3 PL8-6 PL Prep to Process x 3 8 PL8-68 PL Prep to Process x 3 8 PL7-68 PL Prep to Process x 3 PL7-37 PL7-373 Prep to Process x - PL7-3 PL Prep to Process x - PL7-3 PL Prep to Process x PL7-3 PL7-3 PL7-3 PL7-33 Prep to Process x 8 PL7-38 PL Prep to Process x 3 - PL-6 PL Prep to Process x 3 - PL-6 PL Prep to Process x 3 PL-6 PL Prep to Process x 3 8 PL-68 PL Prep to Process x 3 PL-37 PL-373 Prep to Process x PL-3 PL-3 PL7-3 PL7-33 Prep to Process x 8 PL-38 PL Prep to Process x PL- PL-3 PLRP-S Guard Cartridges for x 3 mm, /pk PL6-8 PL6-8 PL6-8 PL6-8 Guard Cartridge holder for 3. x. mm cartridges PL3-6 PL3-6 PL3-6 PL3-6

23 Bulk PLRP-S HPLC Particle Size (μm) Unit PLRP-S Å USP L PLRP-S 3Å USP L PLRP-S Å USP L PLRP-S Å USP L kg PL-6K PL-6K PL-6K g PL-K PL-K PL-K 3 kg PL-67 PL-673 g PL-7 PL-73 - kg PL-6 PL g PL- PL- - kg PL-6 PL-6 g PL- PL- kg PL-6 PL-6 PL-6 PL-63 g PL- PL- PL- PL-3 8 kg PL-68 PL-68 For larger quantities, please contact your local Agilent sales office. AdvanceBio Peptide Mapping Columns Description Part Number.6 x mm,.7 μm x mm,.7 μm x mm,.7 μm x mm,.7 μm x mm,.7 μm mm Fast Guard* mm Fast Guard* mm Fast Guard* 87-9 Maximum Operating Pressure bar 6 bar bar AdvanceBio RP-mAb columns ZORBAX RRHD 3Å.8 μm columns Poroshell 3 columns ZORBAX 3Å 3. and µm columns PLRP-S 3 and µm columns AdvanceBio.7 µm columns * Fast Guards extend column lifetime without slowing down the separation or affecting resolution. Agilent AdvanceBio columns: For faster, more consistent biopharmaceutical analysis Agilent AdvanceBio columns deliver the consistent and exceptional performance you need to separate and characterize peptides and proteins. The science behind the AdvanceBio family improves accuracy, increases productivity, and eliates the interferences that impede your work. What s more, we test AdvanceBio columns rigorously to ensure great results, and back them up with our 6-day full satisfaction warranty. To learn more about perforg high-resolution protein separations with Agilent reversed-phase columns, visit agilent.com/chem/advancebio 3

24 Agilent Biocolumns: Results you can trust for fast, accurate reversed-phase BioHPLC Superior choice and flexibility for reversed-phase biomolecule analysis Cutting-edge Fast LC with advances such as Poroshell technology based AdvanceBio RP-mAb columns for faster analysis and high resolution on any HPLC or UHPLC UHPLC method refinement via ZORBAX RRHD.8 µm columns (stable to bar) Performance, reproducibility, and value proven through millions of injections Fast, consistent, biopharmaceutical analysis: AdvanceBio Peptide Mapping BioHPLC columns let you quickly resolve and identify ao acid modifications in primary structure Exceptional peak shape performance through innovative silica and bonding technologies combine to provide accuracy in protein identity confirmation and impurity analysis Range of selectivities to provide high resolution and recovery for peptides and proteins You also have access to the extensive Agilent applications library for faster method development plus worldwide technical support, speedy problem resolution, and our global infrastructure and delivery network. Load & Lock column hardware for purification Agilent offers Load & Lock prep and process column hardware and packing stations for purification from multi-g to multi-kg quantities of product. The PLRP-S media is available in larger batch sizes to pack these columns. For more information To learn more about Agilent reversed-phase columns, visit agilent.com/chem/advancebio Find an Agilent customer center in your country: agilent.com/chem/contactus Need sample prep for your protein analysis? Agilent Low Protein Binding Filters are the best choice for protein/peptide-related sample filtration, with consistent lowest protein binding during filtration. U.S. and Canada: Europe: Asia Pacific: This information is subject to change without notice. Agilent Technologies, Inc. Printed in the USA November 9, 99-6EN

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