Dedicated UPLC Chemistries for BioTherapeutics Joe Walsh WITS BioPharm Session 2016
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1 Dedicated UPLC Chemistries for BioTherapeutics Joe Walsh WITS BioPharm Session Waters Corporation 1
2 Market Drivers In 2014, 5/10 of the top selling drugs globally were monoclonal antibodies (mabs) (totaling $44B) 1 In 2014, 4/5 of the top selling drugs globally were proteins - 3 mabs 1 Over 50 mab therapeutics have been approved by the FDA/EMA mabs are currently under development 3 8 mabs go off patent between 2013 and At least 4 mabs/year-regulatory approval expected over the next several years GEN Report /approved_mabs.php 3 ecker-2015-the_therapeutic_antibody_marketrprnt.pdf 4 ght/biosimilars-market.asp Waters Corporation 2
3 Biopharmaceuticals vs. Small Molecules Therapeutics Herceptin mab Ibuprofen 2016 Waters Corporation 3
4 Agenda Size Exclusion Chromatography (SEC) Ion Exchange Chromatography (IEX) Novel Column Technologies for Peptide Mapping Glycoprotien Analysis Released N Glycan Analysis Intact Analysis Glycopeptide Mapping Glycan Occupancy 2016 Waters Corporation 4
5 Principles of Size Exclusion Chromatography Separates proteins by their size in solution (Stokes radius) NOT Absolute Molecular Weight Separations are Isocratic Ideally no adsorption to surface of particles Analytes elute in CV 1 resulting in low peak capacities as compared to other methods such as RP methods where analyte elutes in CV 1 Flow 2016 Waters Corporation 5
6 Chromatographic Materials for Peptide and Proteins SEC Cross -Linked agarose (e.g.,ge Healthsciences Sephadex ) Poor mechanical strength Low adsorption of proteins Diol-Bonded, 100% silica (e.g., Tosoh s TSKgel SuperSW mab) Good mechanical properties (strength and pore volume) Residual silanol activity Diol-Bonded, silica-hybrid (Waters BEH SEC HPLC and UPLC) Good mechanical properties (strength and pore volume) Reduced silanol activity Universally applicable 2016 Waters Corporation 6
7 Comparative SEC Column Life (ph 6.8, 150 mm NaCl) AU AU Lysozyme, pki = 10.7 Lysozyme, pi = 10.7 BEH200 SEC shows minimal secondary interactions even after 600 injections Suggestive of DIOL Bleed Suggestive of DIOL Bleed Buffer Peak Minutes Buffer Peak ACQUITY UPLC Protein BEH SEC 200Å, 1.7 µm, 4.6 x 300 mm Injection 19 Injection 618 HPLC 100% Silica-Diol SEC 250Å 4µm 4.6 x 300 mm Injection 19 Injection Minutes 2016 Waters Corporation 7
8 Advantages of UPLC Technology for SEC Separations Requires Columns and Instrumentation to Minimize Band Spreading HPLC Broad Band Broad Peak Less Sensitivity Less Resolving Power Waters UPLC Technology Narrow Peak Increased Sensitivity Increased Resolving Power 2016 Waters Corporation 8
9 UPLC-SEC vs HPLC-SEC of mab monomer and aggregates ACQUITY BEH200 SEC, 1.7 µm 4.6 x 300mm HPLC 100% Silica-Diol SEC 250Å 5µm 7.8 x 300 mm AU AU % Aggregate % Aggregate Minutes 8.00 Minutes Waters Corporation 9
10 Effect of Particle Size /250 Å Comparison 250Å, 5 µm, Silica-Based SEC Å Comparison 450Å, 8 µm, Silica-Based SEC UV Absorbance (280 nm) b 1a 200Å, 3.5 µm, XBridge SEC 5 UV Absorbance (280 nm) b 1a 450Å, 3.5 µm, BEH SEC Minutes Minutes 1a & 1b) thyroglobulin dimer & monomer (1.34 MDa & 669 KDa), 2) IgG (150 KDa), 3) BSA (67 KDa), 4) myoglobin (14 KDa), and uracil (112 Da) Waters Corporation 10
11 Selecting the Optimal Pore Size and Combining Pore Sizes AU ACQUITY UPLC BEH SEC 200Å, 1.7µm (300mm) AU ACQUITY UPLC BEH SEC 200Å, 1.7µm + 450Å, 2.5µm (150mm + 150mm) AU ACQUITY UPLC BEH SEC 450Å, 2.5 µm (300mm) Minutes BEH200 SEC Standard: 1. Thyroglobulin Dimer (1,340 KDa), 2. Thyroglobulin (667 KDa), 3. IgG (150 KDa), 4. BSA (66 KDa), 5. Myoglobin (17 KDa), 6. Uracil (112 Da) Waters Corporation 11
12 Agenda Size Exclusion Chromatography (SEC) Ion Exchange Chromatography (IEX) Novel Column Technologies for Peptide Mapping Glycoprotien Analysis Released N Glycan Analysis Intact Analysis Glycopeptide Mapping Glycan Occupancy 2016 Waters Corporation 12
13 Ion-Exchange Chromatography Cl - Na + Cl - Na + Anion- Exchange Particle Cl - Na + Na + Cl - Anion- Exchange Particle Cl - Na + Cl - Na + Cl - Cl - Cl - Na + Cl - Na + Na + Cl - Elute with Step or Continuous Binds at Low Ionic Strength Gradients of Increasing Ionic Strength Separations are based on net surface charge on protein with oppositely charged groups on ion-exchanger Proteins elute from column using either a gradient of increasing salt concentration (most common) or changing ph (less common) 2016 Waters Corporation 13
14 Ion-Exchange Advantages Moderate resolution Concentrating technique Can load large volumes of a dilute sample Non-denaturing protein elution techniques Preferred first step when a cost-effective affinity purification is not available Limitations Separation by charge and charge distribution; insensitive to other properties For salt gradients, timeconsuming ph optimization required for best resolution. Fractions may need to be desalted prior to the next purification or characterization step (e.g., Mass Spectrometry) 2016 Waters Corporation 14
15 Strong and Weak Cation Exchange Column Comparison 2 3 Weak cation exchanger 0.08 Ribonuclease A cytochrome c 2 AU 0.04 Lysozyme Strong cation exchanger 0.06 AU Minutes Strong and weak cation exchange columns demonstrate different retention 2016 Waters Corporation 15
16 Protein-Pak Hi Res IEX 2016 Waters Corporation 16
17 Attributes of Protein-Pak Hi Res IEX Columns Multi-layered network of ion-exchange groups (SP, CM or Q) o o o Effective diffusion and binding High sample loading and resolution Minimal non-desired interactions No MW limitations: non-porous material QC tested with protein samples for batch-to-batch reproducibility High chemical stability: hydrophilic, polymer-based IEX particles Wide ph range (3-10) high salt concentrations (1M) Standard pressures (up to 1450 psi for CEX and 2175 psi for AEX) Can be cleaned with aggressive washing ecord enabled for data tracking 2016 Waters Corporation 17
18 Waters Auto Blend Plus Technology 12.5% 100mM NaH 2 PO % 100mM Na 2 HPO 4 25mM Sodium Phosphate, 150mM NaCl ph % 1000 mm NaCl 60% H Waters Corporation 18
19 Agenda Size Exclusion Chromatography (SEC) Ion Exchange Chromatography (IEX) Novel Column Technologies for Peptide Mapping Glycoprotien Analysis Released N Glycan Analysis Intact Analysis Glycopeptide Mapping Glycan Occupancy 2016 Waters Corporation 19
20 Reversed Phase Peptide Separations Peptide separations critically important peptide mapping, bottom-up proteomics reversed phase challenges remain LC performance MS performance TFA Ionic analytes peptides Secondary interactions - Poor peak shape Overloading at very low loads (<< neutrals) MS signal suppressing ion pairing agents needed TFA Peptide Still needed: high resolution, high sensitivity peptide separations regardless of eluent additive i.e. formic acid (FA) separations for LC-MS 2016 Waters Corporation 20
21 Competitor s Industry Standard 5 µm Porous Silica C % TFA Ion Pairing Strong A % TFA 0.08% FA % FA Weak not detected Time (min) Competitor s Industry Standard C x 250 mm, Porous 5 µm, 300Å ACQUITY UPLC H-Class Bio 2% ACN for 1 min, then to 50% ACN over 60 min 0.3 ml/min 40 C 214 nm / Xevo G2 QTOF 5.6 µg MassPREP Peptide Mixture 2016 Waters Corporation MassPREP Peptide Mixture Peptide Sequence 1 RASG-1 RGDSPASSKP 2 Angiotensin 1-7 DRVYIHP 3 Bradykinin RPPGFSPFR 4 Angiotensin II DRVYIHPF 5 Angiotensin I DRVYIHPFHL 6 Renin Substrate DRVYIHPFHLLVYS 7 Enolase T35 WLTGPQLADLYHSLMK 8 Enolase T37 YPIVSIEDPFAEDDWEAWSHFFK 9 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ 21
22 Charged Surface Hybrid (CSH) Technology patent pending Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography. P.C. Iraneta, K.D. Wyndham, D.R. McCabe, and T.H. Walter Waters White Paper EN 2011 Expands upon the robust BEH particle technology CSH130 C18 = BEH130 base particle + low level of basic moieties + trifunctional C18/end cap Acidic ph Positive Surface Charge Peptide 2016 Waters Corporation 22
23 MS Signal - FA vs TFA Peak Capacity MS Signal 370 CSH130 C µm 2 P c,4σ % 20% Competitor s SPP Peptide C18 1.7µm BEH130 C µm Competitor s Industry Standard C18 5 µm Fold Decrease in MS Peak Area % TFA % FA 0.10 Percent 0.05 TFA % TFA % FA 0.10 Percent 0.05 TFA Waters Corporation 23
24 Non-Reduced Lys-C Peptide Maps Trastuzumab 1.1 BEH130 C % TFA 0.08% FA Peak Capacity = 207 A % Coverage CSH130 C Peak Capacity = % Coverage A wh (sec) Peak BEH130 C 18 C SH130 C Time (min) 2016 Waters Corporation 24
25 Agenda Size Exclusion Chromatography (SEC) Ion Exchange Chromatography (IEX) Novel Column Technologies for Peptide Mapping Glycoprotien Analysis Released N Glycan Analysis Intact Analysis Glycopeptide Mapping Glycan Occupancy 2016 Waters Corporation 25
26 Glycosylation of Biotherapeutics Fucose GlcNAc Mannose Effector Functions (ADCC/CDC) (fucosylation/galactosylation) Galactose N-acetylneuraminic acid N-glycolylneuraminic acid Low Half Life (high mannose) Anti-Inflammatory (sialylation) Immunogenic (αgal / N-glycolylneuraminic acid) Overall profile sensitive to manufacturing conditions N-glycosylation is a quality attribute of biotherapeutics N-glycan profiles are frequently characterized in detail and routinely monitored Anal Chem 2013, 85 (2), Waters Corporation 26
27 Multiple Strategies Complementary Information Released Glycan Monosaccharide/Sialic Acid Glycopeptide Intact Protein Middle Up/Down HILIC Hydrophilic Interaction Chromatography 2016 Waters Corporation 27
28 Simplified Workflow Conventional 5 Hours to 2 Days Direct Analysis (Organic Solvent Dilution) 10 min 5 min 10 min GlycoWorks RapiFluor-MS N-Glycan Kit Total Sample Prep Time 30 min Patent Pending 2016 Waters Corporation 28
29 Revolutionizing N-Glycan Sample Prep Sensitivity Comparison Fluorescence MS (BPI) RapiFluor-MS Labeled Instant AB Labeled 2-AB Labeled Procainamide Labeled Relative Performance (%) * * (*) Comparative result extrapolated from a published comparison of N-glycans, wherein it was found that procainamide provided comparable fluorescence and up to 50 fold greater ESI-MS sensitivity when compared to 2-AB(Klapoetke et al. 2010) Waters Corporation 29
30 Multiple Strategies Complementary Information Released Glycan Monosaccharide/Sialic Acid Glycopeptide Intact Protein Middle Up/Down HILIC Hydrophilic Interaction Chromatography 2016 Waters Corporation 30
31 Separation of RNase B Glycoforms On Different HILIC Stationary Phases Amide Bonded BEH 300Å Improved Resolution for Large Analytes Highly Branched Glycans 0.4 Intact mabs A Amide Bonded BEH 130Å Unbonded BEH 130Å Time (min) Patent pending 2016 Waters Corporation 31
32 Orthogonality and LC-MS (Glycoforms) Man5 +Man6 +Man7 +Man8 +Man9 Reversed Phase BEH C4 300Å A RNase B aglycosylated Man5 +Man6 HILIC BEH Amide 300Å A 214 RNase B aglycosylated +Man Man7 +Man Time (min) 2016 Waters Corporation 32
33 Orthogonality and LC-MS 0.27 Reversed Phase BEH C4 300Å +Man5 +Man6 +Man7 +Man8 +Man9 A RNase B aglycosylated Man9 Glycoform HILIC BEH Amide 300Å +Man5 +Man A214 RNase B aglycosylated +Man8 +Man Man Time (min) 2016 Waters Corporation m/z Patent pending 33
34 Assaying Deglycosylation and Glycan Occupancy on Intact mabs Intrinsic Fluorescence Detection Trastuzumab Partially Deglycosylated min Trastuzumab Native 2 N-Glycans 1 N-Glycan 21.5% 0 N-Glycans 3.1% 2 N-Glycans 2 N-Glycans 1 N-Glycans Intact 1 N-Glycan 1.0% 0 N-Glycans min Patent pending 2016 Waters Corporation 34
35 Orthogonal Peptide Mapping Techniques Trastuzumab Lys-C Digest 1.0 Peptide BEH C18 300Å 1.7 µm A214 Reversed Phase Non-glycosylated Peptides Peptide Glycoprotein BEH Amide 300Å 1.7 µm HILIC A Glycopeptides Non-glycosylated Peptides Waters Corporation Time (min)
36 Middle-Up Analysis Orthogonality 0.24 Protein BEH C4 300Å 1.7μm Fc/2 Glycoforms 0.18 LC Fd Middle-Up A Time (min) Glycoprotein BEH Amide 300Å 1.7μm LC 0.12 Fd A Fc/2 Glycoforms Time (min) Patent pending 2016 Waters Corporation 36
37 Summary Advanced chromatographic technologies are required for the analysis of complex analytes such as Biopharmaceuticals Size Exclusion Smaller particle size columns provide improved resolution and throughput Physical and chemical column lifetime is improved with hybrid particles (BEH) Ion Exchange Auto Blend Plus Technology allows for change of ph and ionic strengths without preparation of different buffers to simplify methods development Combination of the Protein-Pak Hi Res IEX column benefits and H-Class Bio ACQUITY UPLC o Consistent batch-to-batch performance (tested with protein standards) o Minimal column related carryover o Stable over a wide ph range 2016 Waters Corporation 37
38 Summary cont. Peptide Analysis Novel Charged Surface Hybrid (CSH) Columns provide improved chromatographic performance under MS friendly conditions Balanced peak capacity and MS sensitivity for peptide mapping Glycoprotein Characterization RapiFluorMS can simplify sample preparation and improve MS sensitivity for released N glycan analysis Novel Widepore BEH Amide HILIC column provides high resolution and unique selectivity for analysis of intact glycoproteins, glycoprotein subunits and glycosylated peptides 2016 Waters Corporation 38
39 2016 Waters Corporation 39
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