Thank you for joining us! Our session will begin shortly

Transcription

1 Thank you for joining us! Our session will begin shortly While you are waiting, please feel free to browse our library of program content: Also, click below to learn more about CORTECS, our newest Solid-Core LC Column platform: Waters Corporation 1

2 Friendly Reminders Please use text chat functionality to submit your questions today. Poll Questions Audience participation Providing Live Technical Support during today s event Upon conclusion, follow up information will be available: Recorded version of today s presentation PDF Copy of today s slides Product discount offers Product specific information and reference materials 2014 Waters Corporation 2

3 Today s Speaker Bill Warren has been with Waters Corporation for more than 20 years, having worked in both technical and marketing capacities. He is currently responsible for strategic and tactical implementation of programs that support new bioseparationsproducts and technologies which help accelerate customer productivity in the biopharmaceutical market segment Waters Corporation 3

4 2014 Waters Corporation 4

5 Applications of UPLC Bioseparations 7.5e-2 7.0e-2 6.5e-2 6.0e-2 5.5e-2 HPLC AMQ Lys AU 5.0e-2 4.5e-2 Cys Tyr M et V al Ile Leu Phe 4.0e-2 3.5e-2 3.0e-2 2.5e-2 2.0e-2 1.5e-2 NH3 His S er Arg Gly Asp Glu Thr Ala P ro Deriv Peak 40T 50T 60T 1.0e e-2 8.0e-2 7.0e-2 6.0e-2 UPLC Minutes Amino Acid Analysis UV 260 nm AU 5.0e-2 4.0e-2 3.0e minutes e-2 Time Peptide Mapping G0 2 G0F 3 Man5 4 G0FGN 5 G1 6 G1Fa 7 G1Fb 8 G1FGN 9 Man6 10 G2 11 G2F 12 G1F+SA 13 G2F+SA Oligonucleotides Monoclonal Antibody 0.22 Glycan Analysis BSA Myoglobin Enolase AU ph cytochrome c Phosphorylase b AU AU ph AU ph Protein Reversed Phase Minutes Protein Size Exclusion Minutes Protein Ion Exchange 2014 Waters Corporation 5

6 Topics Customer Requested Attributes for Bioseparation Columns What Goes into Obtaining Improved LC-Based Bioseparations Concerns with UPLC to HPLC Method Transferability Synthetic Oligonucleotide Analyses Amino Acid Analyses Peptide Analyses Protein Analyses Released Glycan Analyses 2014 Waters Corporation 6

7 Customer Requested Attributes for Bioseparation IEX Columns Type Votes Weighted Average Rank Resolution Column-to-column reproducibility Batch-to-batch reproducibility Volume and mass loading capacity Separation speed QC tested with biomolecules (e.g. proteins) Column lifetime Price Non-metallic hardware Matching guard column Other RED: The weighted averages were calculate by: ((#5's*5)+(#4's*4)=(#3's*3)+(#2's*2)+#1's)/5 Particles and System Synergy GREEN: Particles and Manufactury Synergy Source: Waters Web Survey (4,760 contacted / 196 responses) 2014 Waters Corporation 7

8 How to Build Resolution: Efficiency with Selectivity 2014 Waters Corporation 8

9 Particle Size Evolution Early 1970 s 40µ pellicular non-porous coated psi 1000 plates/meter 1m columns 10 min Late 1970 s 10µ Irregular micro-porous psi 25,000 plates/meter 3.9 x 300mm 10 min 1980 s to present day 5 2.5µ spherical micro-porous psi 50,000-80,000 plates/meter 3.9 x 150mm 10 min 2014 Waters Corporation 9

10 Particle Size and Flow Rate van Deemter Equation e eéáöüí=bèìáî~äéåí=íç ç=qüéçêéíáå~ä=mä~íé ebqm H = A term + B term + C term ebqm=======mi^qbp içïéëí=ebqm=z[=léíáãìã=mä~íé=`çìåí iáåé~ê=séäçåáíó a(dp) + b + c(dp) 2 u u _=qéêã EiçåÖáíìÇáå~ä=aáÑÑìëáçåF u ôåãlëéåõ `=qéêã Ej~ëë=íê~åëÑÉêF ^=qéêã Em~êíáÅäÉ=ëáòÉ=~åÇ=Üçï=ïÉää=ÄÉÇ= ï~ë=é~åâéçf ^ÇÇ=íÜÉ=P=íÉêãë=íç=çÄí~áå=Ñáå~ä= î~å=aééãíéê=`ìêîéò 2014 Waters Corporation 10

11 Chromatography Principles Mass Transfer / Diffusion Mobile Phase Analyte Molecules A Adsorption Equilibria B Porous Particle Diffusion-related band broadening C 2014 Waters Corporation 11

12 Smaller Particles The enabler of productivity 2014 Waters Corporation 12

13 Where Does Band Spreading / System Dispersion Occur? Extra Column Within Column Band Spreading: 1) From the Injector 2) Into, through and out of the column 3) Into the Detector 2014 Waters Corporation 13

14 Band Spreading, Peak Height and Resolution LC systems (column and instrument) capable of producing narrower/sharper bands create narrower/sharper peaks This results in better resolution, taller peaks and better sensitivity System with MORE Band Spreading System with LESS Band Spreading Both analytes (blue and red) are not separated [a partial coelution shown as a purple band] Better separation More concentrated Bands Higher Sensitivity 2014 Waters Corporation 14

15 Impact of Band Spreading on Resolution HPLC Broad Band Broad Peak Less Sensitivity Less Resolving Power UPLC Technology Narrow Peak Increased Sensitivity Increased Resolving Power Requires Columns and Instrumentation to Minimize Band Spreading 2014 Waters Corporation 15

16 Sources of Band Spreading Improper Column Connection Resulting Peak Shape Dead / Void VolumeBand Spreading Improper Packed Bed Of Particles Proper No Dead Volume 2014 Waters Corporation 16

17 HPLC vs UPLC SEC Analysis of Insulin Dimer vs Monomer 2014 Waters Corporation 17

18 HPLC vs UPLC Reversed-Phase Gradient Analysis of Derivitized Amino Acids 4 µm Particles 1.8 µm Particles 2014 Waters Corporation 18

19 UltraPerformance LC Technology A class of separation science Based on chromatography columns with very small particles Based on instruments designed and manufactured to take advantage of the small particles Improves resolution, sensitivity, and speed with no compromises to results Suitable for chromatographic applications in general Appropriate for developing new methods Appropriate for improving existing methods SCALEABLE CHEMISTRIES FROM UPLC TO HPLC IF NEEDED 2014 Waters Corporation 19

20 Importance of Method Transferability From Drug Discovery, Development and Manufacturing 2014 Waters Corporation 20

21 Ethylene Bridged Hybrid (BEH) Particles Technology Bridged Ethanes within a silica matrix U.S. Patent No. 6,686,035 B Waters Corporation 21

22 Importance of Batch to Batch and Column to Column Reproducibility Over 30 tests per batch of media totaling over 300 QC response factors 2014 Waters Corporation 22

23 Application-specific Columns ACQUITY UPLC BEH Glycan Column Chemical Tests Chromatographic Test with Biomolecule Standards Individual Column Tests 2014 Waters Corporation 23

24 Manufacturing Consistency ACQUITY UPLC Glycan BEH Amide Columns ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm Quality control testing ensures consistent analyses 2014 Waters Corporation 24

25 UPLC and HPLC-based, 2-AB Labeled Glycan Analyses EU ACQUITY BEH Glycan 1.7 µm 6 UPLC EU x 150 mm 0.50 ml/min XBridge BEH Glycan 2.5 µm XP 4 Alliance HPLC 2.1 x 150 mm 0.34 ml/min P c * half-height = psi (Column, Max) P c * half-height = psi (Column, Max) UPLC-based HPLC-based EU 5.00 XBridge BEH Glycan 3.5 µm Alliance HPLC 2.1 x 150 mm 0.24 ml/min P c * half-height = psi (Column, Max) HPLC-based Minutes 2014 Waters Corporation 25

26 UPLC vs HPLC Relative Abundance Determinations , , um, 0.50 ml/min) um XP, 0.34 ml/min % Abun ndance um, 0.24 ml/min Peak 1 G0-GN Peak 2 G0 Peak 3 G0F Peak 4 Man5 Peak 5 G0FN Peak 6 G1F Peak 7 G1F Peak 8 G1FN Peak 9 Man6 Peak 10 G2 Peak 11 G2F Peak 12 G2FN Peak 13 G1FS1 Peak 14 G2FS1 n=3 Peak 15 A3 Peak 16 A3 *Peaks 8 and µm resolution insufficient, accuracy of the integration is poor 2014 Waters Corporation 26

27 Influence of Extra-Column Band Spreading on Gradient Separations with 1.6um Particles Waters ACQUITY UPLC I-Class N = 18,315 CORTECS UPLC C x 50 mm, 1.6 µm Efficiency (Pla ate Count) Dionex Ultimate 3000 N = 10,974 Waters ACQUITY UPLC H-Class N = 14,072 Agilent 1290 N = 9,392 Shimadzu Nexera N = 6,913 Agilent 1200 N = 6, Waters Alliance 2695 N = 2, Band spreading (µl, 5 sigma) Journal of Chromatography A, 1216 (2009) Waters Corporation 27

28 Gradient offset aligns chromatograms Different instruments and dwell volume No Gradient Offset Programmed Gradient Offset HPLC HPLC * * H-Class Bio Retention Time (min) H-Class Bio Retention Time (min) Sample: Waters MassPrep Pepmix Injection Vol: 95 µl Solvent A: H 2 O with 0.1% TFA Solvent B: MeCN with 0.1% TFA 2014 Waters Corporation 28

29 Peptide Mapping of complex separations Trypsinized Ribonuclease B AU AU A HPLC B * H-Class Bio * Sample: Ribonuclease B Peptidase: Trypsin V5111 Injection Vol: 95µl Solvent A: H 2 O with 0.1% TFA Solvent B: MeCN with 0.1% TFA Relative Retention Time Relative Retention Time Comparison HPLC H-Class Bio Minutes 2014 Waters Corporation Peak Number

30 Peptide Mapping of complex separations Trypsinized Infliximab AU AU HPLC H-Class Bio Sample: Infliximab Peptidase: Trypsin H-Class Bio V5111 Inj. Vol: 95µl Solvent A: H 2 O Solvent B: MeCN Solvent C: 1% TFA Solvent D: H 2 O Retention Time (min) A total of 56 peptide peaks were selected for monitoring Approximately 90 peaks were identified within the chromatographic space Waters Corporation 30

31 UPLC Technology for Synthetic Oligonucleotide Separations 2014 Waters Corporation 31

32 Principles of Ion-Pair, Reversed-Phase Chromatography for Synthetic Oligonucleotides Ion-pairing systems Performance, optimization, MS compatibility Oligonucleotide Separation Technology (OST) Importance of sorbent particle size 2014 Waters Corporation 32

33 Retention Mechanism of Trityl-Off Chromatography Reversed-phase interaction - hydrophobicity of the nucleobases RP with ion-pairing agent - charge-charge interaction (oligo backbone) - hydrophobicity (nucleobases) minutes 10 0 minutes C 18 sorbent TEA + layer on the column surface Waters Corporation 33 - TEA + + -

34 Several Different Ion-Pairing Reagents Available for Synthetic Oligo RP Separations Ion pairing agent buffering acid Abbreviation Triethylammonium acetate TEAA Triethylammonium bicarbonate TEAB Dimetylbutylammonium acetate DMBAA Tributylammonium acetate TBAA Tripropylammonium acetate TPAA Hexylammonium acetate HAA Triethylammonium hexafluoroisopropanol TEA-HFIP 2014 Waters Corporation 34

35 Comparison of TEAA vs. TEA-HFIP for the IP-RP Separation of the same 30mer Oligo Synthesis Mixture UV 260 nm Triethylammonium Acetate Reagent Minutes 32 UV 260 nm Triethylammonium HFIP Reagent T 11 G 12 A A T T G 17 T 18 C 19 C 20 T 21 T 22 T 23 A 24 G 25 G G G C T 30 0 Minutes Waters Corporation 35

36 Agenda Principles of Ion-Pair, Reversed-Phase Chromatography for Synthetic Oligonucleotides Ion-pairing systems Performance, optimization, MS compatibility Oligonucleotide Separation Technology (OST) Importance of sorbent particle size 2014 Waters Corporation 36

37 Importance of Sorbent Particle Size: Smaller C18 particles Yield Improved IP-RP Synthetic Oligo Separations TEA-HFIP, ph 7.9 at 0.2 ml/min at 60 C 5 µm UV 260 nm 3.5 µm 2.5 µm 8 minutes 28 BEH, C18 Particles: 2.1 x 50mm column, 15-60T ladder 2014 Waters Corporation 37

38 Ultimate Separation Performance Obtained using 1.7um Particles with Waters UPLC Technology TEA-HFIP, ph 7.9 at 0.2 ml/min at 60 C 1.7 µm 40T 50T 60T UV 260 nm 2 minutes 11 BEH, C18 Particles: 2.1 x 50mm column, 15-60T ladder 2014 Waters Corporation 38

39 Example of an IP-RP, MS Analysis of a 21 mer Synthetic RNA Mixture 5`-UUC UGU AAU CUC UUG UCU ATT -3` 2014 Waters Corporation 39

40 UPLC Technology for Amino Acid Analysis 2014 Waters Corporation 40

41 Challenges for Amino Acid Analysis Difficult analytical problem Separation o Wide range of properties o Slight differences between pairs o Wide range of matrices Detection o No chromophore o Wide concentration range Analytical method requirements Unequivocal identification, accuracy Precision and linearity Sensitivity and speed Rugged and robust method 2014 Waters Corporation 41

42 Chemistry of AQC Derivatization Reacts readily with both primary and secondary amines Forms stable derivatives Requires no vacuum drying, sample prep or extraction Amendable to automation 2014 Waters Corporation 42

43 HPLC and UPLC Amino Acid Analysis Methods AMQ Asp Ser Glu Gly ILe Leu His NH3 Arg Thr Ala Pro Tyr Cys Val Met Phe Lys HPLC 50 Minute Cycle Time Minutes NH3 Cys Tyr AMQ Met Val NVa His Lys ILe Leu Phe UPLC Ser Arg Gly Asp Glu Thr Ala Pro 10 Minute Cycle Time Waters Corporation Minutes 43

44 UPLC Application Solution for Amino Acid Analysis Turn-key UPLC Amino Acid Analysis application Optimized for the ACQUITY UPLC System Dedicated QC-tested column and reagents Application-specific Performance Qualification Same result day-to-day, instrument-to-instrument, lab-tolab, around the world Target Applications Protein and peptide ID and quantitation Monitoring cell culture media 2014 Waters Corporation 44

45 Solution: UPLC AAA Solution AU AMQ NH3 HyPro His Asn Tau Ser Gln Arg Gly Asp Glu Thr Ala GABA A Pro HyLys1 HyLys2 AABA Orn Deriv Peak Cys Lys Tyr Met Val NVa Ile Leu Phe Trp Minutes 7X improvement in throughput Eliminated need for outsourcing Same day turnaround of samples I was totally amazed that we were able to separate this set of amino acids in nine minutes when, not long ago, it took several hours. VP, Process Development 2014 Waters Corporation 45

46 Amino Acid Analyses of Cell Culture Sample 6 day Gln 3 day Ser Ala Gly Asp Arg Thr Glu 1 day Minutes 2014 Waters Corporation 46

47 UPLC Technology for Peptide Mapping 2014 Waters Corporation 47

48 Ideal Protein Digestion Intact Protein Complete Digestion without generation of modified peptides 2014 Waters Corporation 48

49 The Reality of Many Protein Digestions Intact Protein Complete Digestion without generation of modified peptides Incomplete Digestion Non-specific Cleavages Non-Cleavage Miscleavages Hydrophobic Protein that may have solubility issues 2014 Waters Corporation 49

50 van Deemter Plot 1500da Peptide µl/min 2.1 mm Column 250 µl/min 2.1 mm Column µm (H) Plate Height Increase in chromatographic resolution 1.7 µm Linear Velocity (mm/sec) 2014 Waters Corporation 50

51 Improve Resolution Same Run Time 7.5e-2 7.0e-2 6.5e-2 6.0e-2 5.5e-2 5.0e-2 HPLC 2.1 x 250 mm, 3.5 µm HPLC Peak Capacity = 372 AU 4.5e-2 4.0e-2 3.5e-2 3.0e-2 2.5e-2 2.0e-2 1.5e-2 1.0e e-2 8.0e-2 7.0e-2 6.0e-2 UPLC 2.1 x 150 mm, 1.7 µm UPLC Peak Capacity = 723 AU 5.0e-2 4.0e-2 3.0e-2 2.0e-2 Time Waters Corporation 51

52 Reduce Run Time Comparable Resolution 7.0e-2 6.0e-2 HPLC 2.1 x 250 mm, 3.5 µm 90 min 5.0e-2 AU 4.0e-2 3.0e-2 2.0e-2 1.0e-2 Time e-1 9.0e-2 8.0e-2 UPLC 2.1 x 150 mm, 1.7 µm 55 min 7.0e-2 AU 6.0e-2 5.0e-2 4.0e-2 3.0e-2 2.0e-2 Time Waters Corporation 52

53 Peptide Map Trace Contaminant e AU 1.5e-1 1.0e e-2 * Time 2014 Waters Corporation 53

54 Peptide Map Trace Contaminant 1.4e e-1 1.3e e-1 1.2e e-1 1.1e e-1 1.0e-1 9.5e-2 9.0e-2 8.5e-2 AU 8.0e-2 7.5e-2 7.0e-2 6.5e-2 6.0e-2 5.5e-2 5.0e-2 4.5e-2 4.0e-2 3.5e-2 3.0e-2 2.5e-2 2.0e-2 * Time 2% 1% 0.5% 0.2% 2014 Waters Corporation 54

55 UPLC Technology for Protein Separations 2014 Waters Corporation 55

56 Challenges for Protein Separations Requires the detection of small chemical differences between quite large molecules Employs a variety of analytical techniques that are sensitive to a different property of the proteins Size exclusion for changes in size or aggregation Reversed-phase for detecting a wide range of small changes Ion-exchange for changes in net charge Hydrophobic Regions Carbohydrate Groups Disulfide Linkages Aromatic Groups Net Charge Hydrogen Bonding 2014 Waters Corporation 56

57 HPLC vs UPLC SEC Analysis of Insulin Dimer vs Monomer 2014 Waters Corporation 57

58 UPLC Technology for Glycans Analysis 2014 Waters Corporation 58

59 UPLC vs HPLC Analysis of 2-AB Labeled N-Linked Glycans Protease Digestion Glycoprotein 6 3 Glycan Release Oligosaccharides (Glycans) 6 3 Glycopeptides Monosaccharides 2014 Waters Corporation 59

60 Separation of 2-AB Labeled IgG on ACQUITY UPLC Glycan BEH Amide (HILIC) Column 2014 Waters Corporation 60

61 Summary Holistic approach to separation science UPLC improves resolution, sensitivity, and speed Application specific chemistries Low dispersion system Better separation benefits downstream detection methods o UV or PDA o FLR o Mass Spectrometry UPLC is an innovative technology that can be routinely applied to all aspects of biotherapeutic proteins analysis Synthetic Oligonucleotides Amino Acid Analysis Peptide Separations Intact Proteins Glycan Analysis Waters recognizes need for both UPLC and HPLC Bioseparations Solutions and has offerings and tools to assist in Worldwide Implementation of selected method(s) Waters Corporation 61

62 Thank You for Attending! Post-Event Landing Page 30% Promotional Offer On BioSeparations Columns Full Webinar Recording of Today s Session w/pdf Slide Deck Compilation of TODAY S KEY Literature, Brochures etc For Questions and to Submit your Ideas for our Next Topic Please - mychemrep@waters.com 2014 Waters Corporation 62