Strategies for the Separation and Characterization of Protein Biopharmaceuticals

Transcription

1 Strategies for the Separation and Characterization of Protein Biopharmaceuticals Koen Sandra Webinar in association with SelectScience and Agilent Technologies January 28, 2015

2 Protein biopharmaceuticals Therapeutic macromolecules produced via recombinant DNA technology Used in the treatment of life threatening diseases such as cancer, autoimmune diseases, etc. Global protein therapeutics market: $100 billion (monoclonal antibodies + other recombinant proteins) ±20% of the total pharmaceutical market Within the current decade, more than 50% of new drug approvals will be biologics 2

3 Protein biopharmaceuticals Blockbuster protein biopharmaceuticals: Insulin: Lantus (Sanofi Aventis) EPO: Epogen/Aranesp (Amgen) Trastuzumab: Herceptin (Roche/Genentech) Infliximab: Remicade (J&J) Adalimumab: Humira (AbbVie) Etanercept: Enbrel (Pfizer, Amgen) Several of these blockbusters are, or will soon become, open to the market This has resulted in an explosion of biosimilar activities 3

4 Protein characterization Whether being involved in the development of innovator biopharmaceuticals or biosimilars, an indepth characterization and analysis of these molecules is required during their development and lifetime Analysis is typically more challenging compared to small molecule drugs Proteins are large and heterogeneous 4

5 Protein characterization Typical characteristics Amino acid sequence Amino acid composition Structural integrity Higher order structures Aggregation S S bridges N and O glycosylation N and C terminal sequence Charge variants Deamidation/isomerization Oxidation Clipping N-ter Lc EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTYIHWVRQAPGKGLEW-- Hc C-ter C-ter --NYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK N-ter 5

6 Protein characterization Typical characteristics Amino acid sequence Amino acid composition Structural integrity Higher order structures Aggregation S S bridges N and O glycosylation N and C terminal sequence Charge variants Deamidation/isomerization Oxidation Clipping N-ter Lc EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTYIHWVRQAPGKGLEW-- Hc C-ter C-ter --NYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK N-ter 6

7 Protein characterization Characteristics determined at different levels Protein MW, structural integrity, charge variants, aggregation, modifications Acid hydrolysis Trypsin digestion PNGase F Amino acids Peptides Sugars Amino acid composition Amino acid sequence, modifications, modification sites, disulfide bridges, etc. N glycans 7

8 A wide range of separation modes Charge Size Hydro(phob/phil)icity Affinity CEX, AEX SEC RPLC, HIC, HILIC Affinity Chromatography 8

9 Reversed phase U/HPLC Proteins 9

10 Reversed phase U/HPLC Challenges encountered in RPLC of proteins Issue Reason Solution Peak tailing Secondary ionic interactions High number of pos charges on proteins Peak broadening Low Dm of large molecules Limited access to pores Stationary phase with limited access to residual silanols Ion pairing reagent Higher temperature Widepore phases Higher temperature Efficient stationary phase (sub 2 µm, superficially porous) Adsorption Hydrophobicity Less hydrophobic stationary phases Stronger solvent High temperatures Courtesy of D. Guillarme 10

11 Reversed phase U/HPLC RPLC analysis for identity and purity determination of Herceptin Hc Fab Lc Hc Lc Fab Fc Fc Intact 10 cm x 2.1 mm x 1.8 µm Zorbax 300 SB-C8 Temp: 80 C Flow: 200 µl/min UV: 214 nm Solv A: 0.1% TFA Solv B: 0.1% TFA in ACN %B, 2-25 min 11

12 Reversed phase U/HPLC Mass Spectrometry RPLC-UV-MS of Herceptin Lc and Hc Deconvoluted spectra 12

13 Reversed phase U/HPLC for comparability assessment Lc Hc mau c f Biosimilar mau a b d min e g Originator min 13

14 Reversed phase U/HPLC for comparability assessment 14

15 Reversed phase U/HPLC for comparability assessment G0F + C terminal Lys Undergalactosylation observed in biosimilar 15

16 Widepore Poroshell for comparability assessment x Remicade Advance Bio RP-mAb 5 cm x 4.6 mm x 3.5 µm 450Å C4 Temp: 80 C Flow: 1 ml/min UV: 214 nm Solv A: 0.1% TFA Solv B: 0.1% TFA in 90% ACN %B, 0-6 min 0 x Remicade biosimilar Response Units vs. Acquisition Time (min) x x Remicade Remicade biosimilar Response Units vs. Acquisition Time (min) 16

17 Widepore Poroshell for comparability assessment x Remicade Advance Bio RP-mAb 5 cm x 4.6 mm x 3.5 µm 450Å C4 Temp: 80 C Flow: 1 ml/min UV: 214 nm Solv A: 0.1% TFA Solv B: 0.1% TFA in 90% ACN %B, 0-6 min 0 x Remicade biosimilar x Remicade Remicade biosimilar Response Units vs. Acquisition Time (min) Differences in hydrophobicity due to a 2-point mutation in the AA sequence of the biosimilar Response Units vs. Acquisition Time (min) 17

18 Widepore Poroshell zero carry over x Remicade is a mab which is prone to carry-over on a substantial number of RPLC columns No carry-over observed on Widepore Poroshell column Remicade (4 µg o.c.) Blank Advance Bio RP-mAb 5 cm x 4.6 mm x 3.5 µm 450Å C4 Temp: 80 C Flow: 1 ml/min UV: 214 nm Solv A: 0.1% TFA Solv B: 0.1% TFA in 90% ACN %B, 0-6 min Response Units vs. Acquisition Time (min) 18

19 Widepore Poroshell for comparability assessment Remicade Remicade biosimilar Lc Hc Advance Bio RP-mAb 5 cm x 4.6 mm x 3.5 µm 450Å C4 Temp: 80 C Flow: 1 ml/min UV: 214 nm Solv A: 0.1% TFA Solv B: 0.1% TFA in 90% ACN %B, 0-6 min Differences in hydrophobicity due to a 2-point mutation in the AA sequence of the biosimilar compared 19

20 Ion exchange chromatography Proteins 20

21 Ion exchange chromatography WCX analysis (n=5) of Herceptin highlighting charge variants _ + Na + Na + Na + H 3 N S A G Electrostatic interaction between charged side chains and opposite charged ion exchange functionalities Elution: increase salt concentration or less common ph F Y P + Lys mau pi low (ACIDIC) Asparagine deamidation Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 25 cm x 2.1 mm x 5 µm Agilent Bio-mAb Temp: 30 C Flow: 200 µl/min UV: 214 nm MPA: 10 mm phos ph 7.65 MPB: 10 mm phos ph 7.65, 100 mm NaCl 5-70%B, 0-36 min pi high (BASIC) min 21

22 Ion exchange chromatography mau mau mau mab with one Lc deamidated mab with one Lc deamidated mab with both Lc deamidated mab with both Lc deamidated mab mab mab WCX analysis of stressed and non-stressed Herceptin Non-stressed originator 3 days ph 9 stressed originator min 1 day ph 9 stressed originator min min 22

23 Ion exchange chromatography mau CEX profile of 1 day ph stressed mab CEX fraction collection, reduction using DTT and transfer to RPLC method min Reduced CEX peak 1: RPLC profile N D Reduced CEX peak 2: RPLC profile Lc Hc 23

24 Size exclusion chromatography Proteins 24

25 Size exclusion chromatography SEC analysis of Herceptin highlighting aggregation 30 cm x 4.6 mm x 3 µm Agilent Bio SEC-3 Temp: 24 C Flow: 350 µl/min UV: 214 nm Mobile phase: 150 mm phosphate support mau mab monomer 99.6% Buffer related compound No interaction with surface Separation by means of pores having different accessibility for molecules of different size Elution with solvents that suppress interactions with column packing % mab dimer min 25

26 mau 800 SEC for comparability assessment mab monomer Originator mau 800 mab dimer Buffer related compound Biosimilar min min 26

27 mau 50 SEC for comparability assessment mab monomer Buffer related compound Originator mab dimer mau min Biosimilar min 27

28 Reversed phase U/HPLC Peptides 28

29 The power of peptide mapping Protein measurement is extremely powerful but does not provide the complete picture nor does it allow to localize modifications Which amino acid is glycosylated, oxidized, deamidated, etc.? This can be assessed at peptide level following proteolytic digestion with e.g. trypsin Peptide measurement is also more powerful towards identity/sequence confirmation 29

30 Tryptic peptides Herceptin EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTYIHWVRQAPGKGLEW-- Light Chain B(1-214) Hc Lc Heavy Chain A(1-449) --NYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG 62 identity peptides Modifications Incomplete and aspecific cleavages... > 100 peptides 30

31 Reversed phase U/HPLC: Peptide map C18 F 3 C C O O - + H 3 N Detailed reversed-phase HPLC peptide map for Herceptin identity and purity assessment 25 cm x 2.1 mm x 2.7 µm AdvanceBio C18 Temp: 60 C Flow: 300 µl/min UV: 214 nm Solv A: 0.05% TFA Solv B: 0.05% TFA in ACN 1-45%B, 2-35 min 5 µl (2.4 µg) S silica A G F Y F 3 C C O O - P + Lys Solvophobic interaction between non polar side chains and non polar surface Electrostatic interaction between charged side chains and adsorbed TFA Elution: increase concentration of organic solvent 31

32 Reversed phase U/HPLC: Peptide map * * * Large undigested material T35 T5 T7 T11 T43 T46 T18 T41 T3 T34 T23 T42 T32 T40 T31 T4 T12 T20 T36 T39 T48 T49 * T47 T24 T44 T53 T37-38 T16 T6 T51 T38 T55 T45 + glycans T27 T19 T59 T8 T45 T26 T13 T14 T2 T26 deam T41 ox T29 T50 T62 T22 T54 T1 T3 deam T3 T30 T25 T21 T15 T61 T56 T33 T57 T43 T22-23 T58 T10 T21 pyroglu * Sample preparation related peaks 32

33 LC MS based peptide mapping Compounds extracted out of dataset 33

34 LC MS based peptide mapping Matching of peptides on sequence (MassHunter BioConfirm) Experimental workflow protein peptides Experimental MW Digestion Mass spectrometry In silico workflow Peptide ID protein In-silico digestion (user defined) peptides In-silico modifications (user defined) Theoretical MW 34

35 LC MS based peptide mapping Compounds extracted out of dataset Compounds matched onto protein sequence 35

36 LC MS based peptide mapping Sequence coverage: 98.8% (655 out of 663 amino acids covered) Hc (A-chain) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Lc (B-chain) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 36

37 Reversed phase U/HPLC: Peptide map Originator T45 glycosylated 99,02 % T45 unglycosylated 0,98 % T45 + glycans T27 T16 T6 T19 T2 T47 T24 T51 T37-38 T59 T8 T38 T55 T45 unglycosylated 37

38 Reversed phase U/HPLC: Peptide map T45 glycopeptide separation N ter Hc N ter EEQYNSTYR T45 + G0F Lc C ter C ter T45 + G1Fa T45 + G2F T45 + G1Fb T45 + G0 C ter C ter 38

39 Reversed phase U/HPLC: Peptide map N ter Hc N D N ter T43 Lc N D C ter C ter M Mox T34 T23 T42 T18 T41 T3 T13 T31 T26 + K C ter C ter T25 T33 T14 T50 T1 T15 T21 T29 T22 T26 deam T26 deam T41 ox T62 + K T62 T54 T3 deam T3 T30 T56 T61 39

40 Originator Comparability assessment Biosimilar 40

41 Comparability assessment T45 + G0F Originator T45 + G1Fa T45 + G2F T45 + G1Fb T45 + G0 T45 + G0F Biosimilar Undergalactosylation observed in biosimilar T45 + G1F T45 + G0 41

42 Comparability assessment ASQDVNTAVAWYQQKPGK Originator Biosimilar T3 native 91,75 % 86,92 % T3 deamidation 8,25 % 13,08 % Originator ASQDVDTAVAWYQQKPGK ASQDVNTAVAWYQQKPGK Biosimilar ASQDVDTAVAWYQQKPGK 42

43 The power of mass spectrometry Identification of modification sites Native Deamidation 43

44 Comparability assessment Originator T62 Biosimilar T62 --NHYTQKSLSLSPGK T62+K 44

45 Comprehensive 2D LC (LC x LC) RPLC x RPLC peptide map of Herceptin Analytical and Bioanalytical Chemistry, issue 1,

46 Comprehensive 2D LC (LC x LC) RPLC x RPLC for comparability assessment Originator 1 Originator 2 Clone (biosimilar) 46

47 Comprehensive 2D LC (LC x LC) RPLC x RPLC for comparability assessment Originator 2 MS Originator T62 MS Biosimilar T62 T62+K T62+K 47

48 Hydrophilic interaction chromatography Glycans 48

49 N glycan analysis workflow PNGase F 2-AB labeling LC-FLD (MS) 2-AB Glycan profile 2-AB Clean-up Clean-up 2-Aminobenzamide (2-AB) 49

50 HILIC: 2 AB labeled N glycans Glycan AdvanceBio Glycan Mapping column 15 cm x 2.1 mm x 2.7 or 1.8 µm Temp: 55 C Flow: 400 µl/min Fluorescence detection Solv A: 100 mm NH4-formate ph 4.5 Solv B: ACN 80-60%B, 0-38 min silica Glycan Hydrophilic partitioning between aqueous layer and mobile phase Elution: increase water concentration 50

51 HILIC: 2 AB labeled N glycans LU 3 G0F G1Fa G1Fb 1.8 µm fully porous particles G0-GlcNAc G0F-GlcNAc G0 G1-GlcNAc G1F-GlcNAc G1a Man5 G1b G2F LU G0-GlcNAc G0F-GlcNAc G0 G1-GlcNAc G0F G1F-GlcNAc Man5 G1a G1Fa G1Fb min 2.7 µm superficially porous particles G2F min 51

52 The power of mass spectrometry MS/MS spectrum of 2AB labeled G0F AB AB AB AB AB AB AB AB AB AB AB AB AB AB AB 52

53 Comparability assessment LU LU G0F G0 G1Fa G1Fb G2F Originator min Biosimilar min Undergalactosylation observed in biosimilar 53

54 Biosimilar Cell culture optimization Bringing glycosylation within originator specifications by adding uridine, galactose and manganese to the CHO growth medium LU LU LU LU LU G0 G0F G1F G2F Biosimilar Biosimilar: 4x Biosimilar: 8x Biosimilar: 16x Biosimilar: 24x min min min min min 54

55 Biosimilar Cell culture optimization Bringing glycosylation within originator specifications by adding uridine, galactose and manganese to the CHO growth medium Glycan Biosimilar 1.8 µm fully porous HILIC particles Biosimilar 4x Biosimilar 8x Relative intensity Biosimilar 16x Biosimilar 24x Specifications originators % G0 GlcNAC / 0.09 % G0F GlcNAc / 0.87 % G / 0.29 % G1 GlcNAc / 0.34 % G0F / 6.47 % Man / 0.35 % G1a + % G1F GlcNAc / 0.30 % G1b / 0.20 % G1Fa / 3.48 % G1Fb / 0.89 % G1F / 4.38 % G2F /

56 Acknowledgement Isabel Vandenheede, Emmie Dumont and Pat Sandra (RIC, Kortrijk, Belgium) The colleagues from the biopharmaceutical industry who trigger and inspire us in developing and applying chromatographic and mass spectrometric methodologies and strategies to tackle their challenging requests Maureen Joseph, Gina Goggings, Linda Lloyd, Phu Duong (Agilent Technologies, Wilmington, Delaware) 56

57 Strategies for the Separation and Characterization of Protein Biopharmaceuticals Improved LC Column Choices for Bioseparations January 28, 2015 Confidentiality Label February 12,

58 Strategies of Column Selection for Separation and Characterization of Protein Biopharmaceuticals Reversed-phase LC Issues and Solutions Issue Challenge Reason Solution Peak tailing Lower resolution Less accuracy Secondary ionic interactions High number of pos charges on proteins Stationary phase with limited access to residual silanols Ion-pairing reagent Higher temperature Peak broadening Lower resolution Reduced sensitivity Adsorption Poor recovery Less sensitivity Low Dm of large molecules Limited access to pores Widepore phases Higher temperature Efficient stationary phase (sub 2 µm, superficially porous) Hydrophobicity Less hydrophobic stationary phases Stronger solvent High temperatures Column parameters are important to solve problems of efficiency/resolution and recovery for improved LC and LC/MS characterization of proteins. Confidentiality Label February 12,

59 Reversed Phase Column Choices Improve Efficiency/Resolution and Recovery AdvanceBio RP-mAB The optimum high speed, large molecule resolution for use with both HPLC and UHPLC systems Superficially porous, 3.5um particle 0.25 um 3.0 um 450Å pores The most popular phases for proteins, plus a unique selectivity. C4, C8, Diphenyl 3.5 um Superficially porous particle ZORBAX RRHD 300Å, 1.8um Fast, high resolution UHPLC analysis of proteins, including intact mabs, and protein fragments Totally porous 1.8um silica particles with 300Å pores 1200 bar for UHPLC use Suitable for intact, fragments and digests The most popular phases for proteins plus a unique selectivity. SB-C3, SB-C8, SB-C18, Diphenyl StableBond bonding for long lifetime with TFA ion-pairing reagent Confidentiality Label February 12,

60 Fast Intact mab Analysis and Comparison of Phases Short 3 minute separation, with each phase unique. mau DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_ \ D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_ \ D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_ \DIP D) AdvanceBio RP-mAb C4 AdvanceBio RP-mAb SB-C8 AdvanceBio RP-mAb Diphenyl DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_ \ D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_ \ D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_ \DIP D) mau min Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water/ipa (98/2) Mobile phase B: IPA/acetonitrile/MPA (70/20/10) Flow rate: 1.0 ml/min Gradient: 10-58% B in 4 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 5 μl injection of Humanized Recombinant Herceptin Variant IgG1 Intact from Creative Biolabs (1 mg/ml) Temperature: 80 C Detection: 254nm min Agilent Technologies February 12,

61 Reversed Phase Peptide Mapping Resolution Maximized with SPP AdvanceBio Peptide Mapping UHPLC column efficiency and resolution for your complete peptide map HPLC and UHPLC 2.7um superficially porous particle LC and LC/MS Minimal peak tailing Optimal C18 bonding 0.50 um 8 x x min. Analysis 0.2mL/min 140 Bar Acquisition Time (min) 14 min. Analysis 0.6mL/min 433 Bar TIC 2.1 x 100mm, 2.7um 1.7um 2.7um Å pores Acquisition Time (min) Confidentiality Label February 12,

62 Choices for Other Modes of Chromatography - SEC SEC for aggregation analysis Columns are porous silica with typical lengths of 250 or 300 mm Typical flow rate is 1.0 ml/min on a 7.8 mm ID column or 0.35 ml/min on a 4.6 mm ID column To increase resolution (through increased pore volume) run columns in series (costs time!) To increase resolution, use smaller particle sizes (3um) To reduce secondary interactions and improve recovery maximize inertness BioSEC-3, 7.8 x 300mm, 3um 3 m particle Proprietary hydrophilic coating to prevent secondary interaction 100Å, 150Å, 300Å pore sizes High efficiency and resolution Faster SEC separations Can be run with low salt buffers Confidentiality Label February 12,

63 Improved SEC Efficiency With Smaller Particles Peak Protein Efficiency Gain SEC-3, 300Å (7.8x300mm) SEC-5, 300Å (7.8x300mm) 1 Thyroglobulin 2.2X BSA Dimer 1.9X BSA 2.0X Ribonuclease A 2.0X Uracil 1.4X Agilent Bio SEC-3, 300Å, 7.8 x 300 mm 93 bar Agilent Bio SEC-5, 300Å, 7.8 x 300 mm 45 bar Min Column: Bio SEC-3 300Å and Bio SEC-5 300Å Buffer: 150 mm Phosphate buffer, ph 7 Flow rate: 1.0 ml/min for 7.8 x 300 mm Temperature: Ambient (~23 C) Detection: UV 214nm Injection: 10 µl (3 L for 4.6 x 300 mm) Sample: 1) Thyroglobulin (1.0 mg/ml), 670 kd; 2) BSA dimer, 132 kd; 3) BSA (1.0 mg/ml), 66 kd; 4) Ribonuclease A (1.0 mg/ml), 13.7 kd, and 5) Uracil (2.5 g/ml), 120 D.

64 Choices for Other Modes of Chromatography - IEX IEX for charge variants Typically non-porous 10um polymeric particles, 250mm long Separation with high salt or ph changes Non-specific interactions with surfaces can reduce resolution Metal ions eluting from instrument can lower resolution and cause column poisoning To increase resolution choose smaller particle sizes (5 and 3um) To maximize recovery choose inert systems BioMAb 3 or 5um 4.6 mm Non-porous PS/DVB particles Uniform polymeric hydrophilic coating and WCX layer, specifically designed for antibody separations with minimal non-specific interactions Fully Bio-inert choices for maximum recovery - 10 µm, 5 µm with PEEK Also available in 3 µm and 1.7 µm particle sizes for highest resolution Confidentiality Label February 12,

65 Improving Ion Exchange Chromatography (IEX) Resolve charge variants with BioInert LC and column. mau % B in 30min min Better peak shape and higher efficiency were achieved with a smaller particle size on the Agilent Bio MAb 5 vs. Bio MAb 10 column. Incomplete separation due to protein/ system interactions. Massive fronting and tailing makes quantitation impossible. 9 Characterization of mab 2/12/2015

66 Choices for Other Modes of Chromatography HILIC Released glycan analysis by HPLC-FLD 1260 Infinity Bio-inert HPLC/FLD + AdvanceBio Glycan Mapping, 2.7 um (superficially porous) Why Amide HILIC? Selectivity is stable over column lifetime High peak capacity RT increases with size, depending on monosaccharide type and position + AdvanceBio Glycan Mapping, 1.8 um 1290 Infinity UHPLC (totally porous) 10

67 Columns can Reduce Challenges in Chromatography for the Characterization of Monoclonal Antibodies Titer determination and purification Affinity Chromatography Protein identification and impurity profiling Reversed-phase chromatography (RP) Glycan analysis Hydrophilic interaction chromatography (HILIC) Charge variant analysis Ion exchange chromatography (IEX) Aggregation analysis Size exclusion chromatography (SEC) Agilent Technologies February 12,