'Tips and Tricks' for Biopharmaceutical Characterization using SEC
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1 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation 2012 Waters Corporation 1
2 Waters Commitment To develop, commercialize and market columns that, when used on Waters ACQUITY UPLC systems, provide speed, sensitivity, resolution, and reproducibility not previously achieved for characterization of biological macromolecules by traditional HPLC Waters Corporation 2
3 Liquid Chromatography Protein Separation Modes Protein Structure Primary, Secondary, Tertiary Structure Carbohydrate Groups Net Charge Hydrophilic Groups Hydrophobic Regions Aromatic Groups Disulfide Linkages Hydrogen Bonding 2012 Waters Corporation 3
4 Liquid Chromatography Protein Separation Modes Protein Structure Primary, Secondary, Tertiary Structure Carbohydrate Groups Net Charge Hydrophilic Groups Hydrophobic Regions Aromatic Groups Disulfide Linkages Hydrogen Bonding 2012 Waters Corporation 4
5 Agenda Size-Exclusion Chromatography Theory and practice ACQUITY UPLC Columns for SEC o ACQUITY BEH450 SEC, 2.5 µm Columns o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7um Columns Monoclonal Antibody Application o SEC-MS Applications Insulin and Small Protein Applications Factors Influencing Component Resolution Considerations to extending column life 2012 Waters Corporation 5
6 Principles of Size Exclusion Chromatography of Proteins Separates proteins by their size in solution (Stokes radius) Separations are Isocratic Tends to be used as a Polishing isolation step or as an analytical technique to determine presence of protein aggregates Generally a lower resolving technique compared to other methods such as ion-exchange or reversed-phase methods 2012 Waters Corporation 6
7 Size Exclusion Chromatography No adsorption to surface of particles Large molecules elute before small molecules Large molecules cannot access pores Small molecules access pores within particle dimer monomer 2012 Waters Corporation 7
8 Common SEC applications: Biotherapeutics Types Monoclonal Antibodies Antibody Conjugates Fc Fusion Proteins Synthetic Oligonucleotides Protein Subunit Vaccines Recombinant Proteins 2012 Waters Corporation 8
9 Agenda Size-Exclusion Chromatography Theory and practice ACQUITY UPLC Columns for SEC o ACQUITY BEH450 SEC, 2.5 µm Columns o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7um Columns Monoclonal Antibody Application o SEC-MS Applications Insulin and Small Protein Applications Factors Influencing Component Resolution Considerations to extending column life 2012 Waters Corporation 9
10 UPLC Systems for Biopharmaceutical Analysis Wide range of applications Complete Solutions Instrumentation o o o o UPLC System (s) ACQUITY UPLC System ACQUITY UPLC H-Class System ACQUITY UPLC H-Class Bio System UV, FLR, PDA and MS Detections Application Specific Chemistries Developed and designed with applications QC Tested with application Optimized for UPLC Software Data Analysis Information management Focused on customer application requirements 2012 Waters Corporation 10
11 Advantages of UPLC Technology for SEC Separations Columns and Instruments both Minimize Band Spreading HPLC Broad Band Broad Peak Less Sensitivity Less Resolving Power Waters UPLC Technology Narrow Peak Increased Sensitivity Increased Resolving Power 2012 Waters Corporation 11
12 Effect of System Dispersion on ACQUITY UPLC BEH200 SEC 1.7 µm separation USP Res= 2.37 UP-SEC ACQUITY UPLC BEH200 SEC 1.7 µm 4.6 x 300mm USP Res= 1.37 HP-SEC ACQUITY UPLC BEH200 SEC 1.7 µm 4.6 x 300mm Large system dispersion decreases resolution Sample: Human polycolonal IgG 2012 Waters Corporation 12
13 ACQUITY UPLC BEH200 and BEH125 SEC 1.7 µm Columns, and BEH450 SEC 2.5 µm Columns Application Areas Molecular weight ranges dependent on pore size: o BEH450: 100,000 to 1,500,000 Daltons o BEH200: 10,000 to 450,000 Daltons o BEH125: 1,000 to 80,000 Daltons Determination of protein / peptide molecular weight Quantifying protein / peptide aggregates primarily in therapeutic monoclonal antibodies, EPO, and Insulin Determination of size heterogeneity in a sample 2012 Waters Corporation 13
14 BEH Technology Particles Bridged EthylSiloxane/Silica Hybrid EtO 4 Si EtO EtO OEt Tetraethoxysilane (TEOS) EtO EtO + Si 1 EtO CH 2 CH 2 Si OEt OEt OEt Bis(triethoxysilyl)ethane (BTEE) EtO EtO Si Si CH 2 CH 2 Si O O O Si OEt OEt O O Si Si O Polyethoxysilane (BPEOS) OEt OEt O O Et Et n Bridged Ethanes In Silica Matrix U.S. Patent No. 6,686,035 B2 Anal. Chem. 2003, 75, Waters Corporation 14
15 HPLC to UPLC SEC Comparison ACQUITY UPLC BEH200 SEC,1.7 µm 4.6 x 300mm HPLC 100% Silica-Diol SEC 250Å 5µm 7.8 x 300 mm % Aggregate % Aggregate Murine monoclonal antibody - Scaled load Conditions: 0.4 ml/min; 25mM Sodium Phosphate, ph 6.8, 0.15 M NaCl 2012 Waters Corporation 15
16 Calibration Curves of ACQUITY UPLC BEH450, BEH200, and BEH125 SEC Columns 2012 Waters Corporation 16
17 Protein Adsorption and Size- Exclusion Chromatography Proteins can interact or adsorb onto the SEC packing material These interactions create undesired and unpredictable retention of proteins (i.e. proteins not separated by size in solution) SEC particles frequently coated with a hydrophilic reagent to minimize non-desired ionic interactions between proteins and packing material Mobile phase additives (e.g., 150mM NaCl) may decrease nondesired ionic interactions between proteins and packing material 2012 Waters Corporation 17
18 BEH SEC Particle Overview The packing material is based on our patented Bridged Ethyl Hybrid base particle and effective diol bonding, which provide a stable chemistry with minimal secondary interactions Waters Corporation 18
19 Comparative SEC Column Life Lysozyme, pki Lysozyme, = 10.7 pi = 10.7 Suggestive of DIOL Bleed Suggestive of DIOL Bleed HPLC 100% Silica-Diol SEC 250Å 4µm 4.6 x 300 mm Injection 19 Injection Waters Corporation 19
20 Comparative SEC Column Life Lysozyme, pki Lysozyme, = 10.7 pi = 10.7 Suggestive of DIOL Bleed Suggestive of DIOL Bleed HPLC 100% Silica-Diol SEC 250Å 4µm 4.6 x 300 mm Injection 19 Injection ACQUITY BEH200 SEC, 1.7 µm 4.6 x 150 mm Injection 19 Injection BEH200 shows minimal secondary interactions even after 600 injections 2012 Waters Corporation 20
21 Column Stability Log Mw Calibration Curve 0 hours 12 hours 24 hours 36 hours 48 hours 60 hours Protein MW Thyroglobulin Ferritin Aldolase BSA Ovalbumin Carbonic Anhydrase Ribonuclease A Aprotinin 6500 Uracil Elution volume (ml) Protein standard analyzed over 48 hours Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, ph 6.8; 280 nm Elution volume for all proteins within 0.2% RSD 2012 Waters Corporation 21
22 Influence of Ionic Strength on Peak Shape and Retention Conventional 100% Silica-Diol Coated SEC Column 4.6 x 300 mm lysozyme 10 mm lysozyme 25 mm lysozyme 100 mm Flow rate: 0.5 ml/min; Mobile phase: 10, 25 or 100 mm sodium phosphate, ph Waters Corporation 22
23 Influence of Ionic Strength on Peak Shape and Retention lysozyme ACQUITY BEH200 SEC 1.7 µm column, 4.6 x 150mm 10 mm mm mm Flow rate: 0.5 ml/min; Mobile phase: 10, 25 or 100 mm sodium phosphate, ph Waters Corporation 23
24 BEH125 SEC, 1.7um Batch-to to-batch Reproducibility Batch 1 Analyte pl MW 1. Thyroglobulin, 0.1 mg/ml , Ovalbumin, 0.3 mg/ml , Ribonuclease A, 0.3 mg/ml , Uracil, 0.05 mg/ml N/A Batch 2 BEH125 SEC Protein Standard Part No Batch Conditions: 100mM Sodium Phosphate ph 6.8; 0.3 ml/min; 30 C; 4.6x150mm 2012 Waters Corporation 24
25 BEH200 SEC, 1.7um Batch-to to-batch Reproducibility BEH200 SEC Protein Standard Part No Waters Corporation 25
26 Combining Pore Sizes for Added Method Development Flexibility ACQUITY BEH200 SEC (300mm) BEH450 SEC Protein Standard Part No ACQUITY BEH200 SEC and BEH450 SEC (150mm + 150mm) ACQUITY BEH450 SEC (300mm) Waters Corporation 26 Compounds: 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)
27 Column Reproducibility 0.05 Batch 1, Column Batch 1, Column Batch 1, Column Batch 2, Column Batch 2, Column Humanized monoclonal antibody Conditions: 25mM Sodium Phosphate, ph 6.8, 0.15 M Sodium Chloride, 0.4 ml/min Retention times within 0.2min 2012 Waters Corporation 27
28 Agenda Size-Exclusion Chromatography Theory and practice ACQUITY UPLC Columns for SEC o ACQUITY BEH450 SEC, 2.5 µm Columns o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7um Columns Monoclonal Antibody Application o SEC-MS Applications Insulin and Small Protein Applications Factors Influencing Component Resolution Considerations to extending column life 2012 Waters Corporation 28
29 Monoclonal Antibody Adapted from Alain Beck Center of Immunology 2012 Waters Corporation 29
30 Protein Structure N Native U Unfolded or I Intermediate Soluble Aggregates Insoluble Aggregates Addressing particulate and aggregation issues of therapeutic protein products, Shi, L, PEGS, May 2011 Soluble aggregates and insoluble particles may affect immunogenicity and efficacy of biotherapeutic 2012 Waters Corporation 30
31 Orthogonal Techniques for Characterization Dynamic Light Scattering C FFF-MALS Static Light Scattering SEC Counter principle Flow Imaging Microscopy Light Microscopy Visual Inspection Microscope nm µm mm cm monomers oligomers Aggregates subvisible particles Particles Visible particles E. Freud, PDA Visual Inspection Forum, Oct Waters Corporation 31
32 Column Lifetime Dimer = 0.46% USP Res = 2.35 Injection mab Dimer = 0.49% USP Res = 2.27 Injection mab Humanized monoclonal antibody Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, ph 6.8; 280 nm Column: 4.6 x 300 mm 2012 Waters Corporation 32
33 Effect of Flow Rate ml/min % Aggregate Flow Rate (ml/min) Average Std Dev % RSD ml/min ml/min Triplicate injections overlaid No observable trend in aggregation with flow rate Murine monoclonal antibody (63 µg load) 2012 Waters Corporation 33
34 Effect of Particle Size: Analysis of LMW Species mab dimer mab LMW peak LMW species BEH200 SEC 1.7µm Å, 4µm HPLC 100% Silica-Diol Å, 5µm HPLC 100% Silica-Diol Å, 5µm HPLC 100% Silica-Diol Humanized monoclonal antibody biotherapeutic Conditions: 25 mm Sodium Phosphate, 0.15 M Sodium Chloride Flow rates and Injection volumes scaled for column dimensions 2012 Waters Corporation 34
35 Agenda Size-Exclusion Chromatography Theory and practice ACQUITY UPLC Columns for SEC o ACQUITY BEH450 SEC, 2.5 µm Columns o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7um Columns Monoclonal Antibody Application o SEC-MS Applications Insulin and Small Protein Applications Factors Influencing Component Resolution Considerations to extending column life 2012 Waters Corporation 35
36 Precautions in SEC-MS Protein structure in solution depends on ph Ionic strength Buffer and salt Additives Good ionization conditions are different from conditions for biological activity Validation required when buffer is changed Special uses are valuable Fast desalting Clips 2012 Waters Corporation 36
37 LC/MS Compatible Mobile Phase on ACQUITY UPLC BEH200, SEC, 1.7um 0.20 PBS mM Ammonium Formate Similar retention time/ peak shape observed with MS compatible mobile phases 2012 Waters Corporation 37
38 Humanized Monoclonal Antibody: MS Compatible/Native Mobile Phase 100mM Ammonium Formate e e e e e e e PBS Time Flow Rates: 100mM Ammonium Formate mL/min, PBS- 0.4 ml/min Lower flow rate for MS compatibility 2012 Waters Corporation 38
39 SEC-MS Humanized Monoclonal Antibody 1.8e : Diode Array Da Range: 6.757e-1 1.6e-2 1.4e-2 1.2e-2 1.0e e e-3 4.0e e : TOF MS ES TIC 7.58e TIC % MS: Xevo G2 Q Tof Conditions: 100mM Ammonium Formate, Flow rate: 0.15 ml/min Post UV detection additive: ACN, 0.8% Formic acid Scan 2012 Waters Corporation 39
40 Extracted Spectrum Herceptin 50%ACN,.4% FA_100mm Amm Form_0.15 ml/min_40cv_autoqua 7Oct11_PH_SEC_BEH200_Ext_T_ACN_pt8FA_2_5 907 (15.351) Sm (SG, 10x5.00); Sb (15,2.00 ); Cm (891:932) % : TOF MS ES+ 4.34e3 Intact IgG MW 148,221 Peak 1 0 m/z Oct11_PH_SEC_BEH200_Ext_T_ACN_pt8FA_2_5 982 (16.619) Sm (SG, 10x5.00); Cm (969:996) 1: TOF MS ES e3 % Clip MW 100,764 Peak 2 0 m/z Oct11_PH_SEC_BEH200_Ext_T_ACN_pt8FA_2_ (19.493) Sm (SG, 10x5.00); Cm (1116:1184) 1: TOF MS ES e % Low MW Species Peak m/z Deconvoluted molecular weight determined using MaxEnt Waters Corporation 40
41 SEC-UV UV-MS: A generic methodology for screening intact and reduced antibodies HC Desalting LC/HC Resolution Detect Clips No Sample Concentration required LC UV 280 HC-HC HC Mass Spectrum HC LC Mass Spectrum TIC HC-HC LC Conditions: System, ACQUITY UPLC TM with TUV optical detector and Synapt G2 QTof MS Flow Rate: 0.2 ml/min 0.1%TFA and 0.1%FA in 30% ACN 2012 Waters Corporation 41
42 Agenda Size-Exclusion Chromatography Theory and practice ACQUITY UPLC Columns for SEC o ACQUITY BEH450 SEC, 2.5 µm Columns o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7um Columns Monoclonal Antibody Application o SEC-MS Applications Insulin and Small Protein Applications Factors Influencing Component Resolution Considerations to extending column life 2012 Waters Corporation 42
43 Resolution of Proteins and Peptides (Aqueous) A ACQUITY UPLC BEH125 SEC 1.7um 4.6 x 300mm BioSuite125 UHR SEC 4.6 x 300mm Carb. Carb. Anhyd. Anhyd. (29,000) (29,000) Myoglobin Myoglobin (16,900) (16,900) Ubiquitin Ubiquitin (8,565) (8,565) RASG RASG (898) (898) G-G-G G-G-G (189) (189) G-G (132) (132) Uracil Uracil (112) (112) BSA (66,000) (66,000) Ovalbumin Ovalbumin (44,000) (44,000) Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, ph 6.8, 0.4 ml/min - - BEH125 column provides increased resolution throughout the lower end of the peptide mass range (132 29,000) Waters Corporation 43
44 ACQUITY UPLC BEH125 SEC 1.7µm Column Reproducibility % RSD Retention Time Range %RSD/Retention Time Range G-G G-G-G RASG Aprotinin Ubiquitin Cytochrome C Myoglobin Carb. Anhyd. Ovalbumin BSA BSA Dimer Insulin Monomer Insulin Dimer Peptide/Protein Table 1. Retention time reproducibility for 5 ACQUITY UPLC BEH125 SEC 1.7 µm columns (4.6 mm x 30cm) using aqueous and organic (insulin separation method only) mobile phases Waters Corporation 44
45 Resolution of Small Protein BioSuite 125 4µm UHR 4.6 x 300 mm column ACQUITY UPLC BEH125 SEC 1.7µm 4.6 x 300 mm column USP Rs= USP Rs= Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, ph 6.8, 0.4 ml/min, sample 2 mg/ml USP monomer/aggregate resolution was 1.7 times greater on the BEH µm SEC column as compared to 4 µm pore diol-coated silica column Waters Corporation 45
46 HPLC/UPLC Column Comparison ACQUITY UPLC BEH µm (4.6 x 300 mm) Rs = 3.7 USP Plate Count = 15K Flow Rate = 0.4 ml/min Rs = 2.1 USP Plate Count = 3K Flow Rate = 0.5 ml/min HMWP 10µm (7.8 x 300 mm) Conditions: Mobile Phase: L-arginine (1.0 g/l) /acetic. acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm, Injection volume: (Waters HMWP) tested to perform in the European Pharmacopoeial method. Increase in HMW resolution observed in shorter run-times 2012 Waters Corporation 46
47 BEH125 SEC, 1.7um Column Life Insulin Analysis Injection 26 ACQUITY UPLC BEH125, SEC 1.7µm 4.6 x 300 mm Injection Comparable absolute retention time change observed for both columns Conditions: Mobile Phase: L-arginine (1.0 g/l) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm, Sample: Human Insulin ( 4mg/mL), Injection volume: 5 µl 2012 Waters Corporation 47
48 Column Stability for Insulin Analysis Retention Time USP Resolution Retention Time (min) USP Resolution Injection Number Over 800 injections the retention time of the insulin monomer peak and the resolution between insulin monomer and dimer peaks are maintained Waters Corporation 48
49 Effect of Pore Size: Insulin ACQUITY UPLC BEH125 SEC 1.7 µm ACQUITY UPLC BEH200 SEC 1. 7µm Control Control Sample 1 Fragment Sample Sample 2 Sample Conditions: Mobile Phase: L-arginine (1.0 g/l) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm The HMW and insulin fragments are better resolved on the 125Å pore diameter column as compared to the 200Å pore diameter 2012 Waters Corporation 49
50 Effect of Particle Size: Insulin ACQUITY UPLC BEH125, 1.7µm 4.6 x 300mm Control Rs= 3.37 HMW BioSuite125 UHR, 4µm 4.6 x 300mm Control Rs= 2.21 Control Rs= 2.08 Insulin HMWP, 10µm 7.8 x 300mm Sample 1 Rs= 2.63 Fragment Sample 1 Rs= 1.93 Sample 1 Rs= Sample 2 Rs= 2.63 Sample 2 Rs= 1.92 Sample 2 Rs= Improved resolution of HMW and Fragment peaks observed with BEH um column Conditions: Mobile Phase: L-arginine (1.0 g/l) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm, Column dimensions: 4.6 x 300mm BEH125 and BioSuite125, 7.8 x 300mm Insulin HMWP, Flow rate: 0.4 ml/min (HMWP: 0.5 ml/min), Injection volumes: scaled 2012 Waters Corporation 50
51 Agenda Size-Exclusion Chromatography Theory and practice ACQUITY UPLC Columns for SEC o ACQUITY BEH450 SEC, 2.5 µm Columns o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7um Columns Monoclonal Antibody Application o SEC-MS Applications Insulin and Small Protein Applications Factors Influencing Component Resolution Considerations to extending column life 2012 Waters Corporation 51
52 Factors Influencing Resolution Resolution increases with lower injection volumes Resolution increases with lower flow rate Ideal flow rate is lower than typically running, however will sacrifice speed Resolution increases with column length Baseline resolution typically achieved at 50%-100% molecular weight difference 2012 Waters Corporation 52
53 Loading Capacity: Undiluted Monoclonal Antibody Biopharmaceutical 2.60 Injection Volume, Total Load µl, 300µg Aggregates 21.8% 10 µl, 200µg % 21.0% 5 µl, 100µg Humanized IgG (20 mg/ml), 4.6 x 150 mm column Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, ph 6.8; 280 nm 2012 Waters Corporation 53
54 Effect of Column Length: Monoclonal Antibody USP Res= % 98.88% 150 mm aggregates mab USP Res= % 98.76% 300 mm aggregates mab Murine monoclonal antibody (load: 6.4 µg - 150mm; 12.7 µg -300mm) Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, ph 6.8;214 nm 2012 Waters Corporation 54
55 Effect of Flow Rate on Rs ml/min Rs= 2.4 ~1500 psi dimer IgG ml/min Rs= 1.8 ~3000 psi ml/min Rs= 1.3 ~6000 psi Resolution increases with lower flow rates Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, ph 6.8;280 nm Column: BEH200 SEC 1.7 µm, 4.6 x 150mm 2012 Waters Corporation 55
56 Effect of Sample Load : Myoglobin Effect of Volume Load 1.90 Effect of Concentration Injection Volume USP Res Concentration (mg/ ml) USP Res Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, ph 6.8, 0.4 ml/min, sample 2 mg/ml, Column: ACQUITY UPLC BEH µm SEC, 4.6 x 300 mm column Myoglobin: 5 mg/ml (volume load, and 20 ul injection volume (concentration) Increased injection volumes can result in a significant loss of resolution in UPLC-SEC analyses Waters Corporation 56
57 Effect of Salt Anion on BEH200 SEC, 1.7um 4.6 x 150mm Peak Shape Different anions of sodium salt additive Buffer: 10mM sodium phosphate, ph 6.8 and 200mM of additive ( unless otherwise noted) Sample: Thyroglobulin, IgG, BSA, Myoglobin, Uracil 2012 Waters Corporation 57
58 Effect of Salt Cation on BEH200 SEC, 1.7um 4.6 x 150mm Peak Shape Different cations of chloride salt additive Buffer: 10mM sodium phosphate, ph 6.8 and 200mM of additive Sample: Thyroglobulin, IgG, BSA, Myoglobin, Uracil 2012 Waters Corporation 58
59 Agenda Size-Exclusion Chromatography Theory and practice ACQUITY UPLC Columns for SEC o ACQUITY BEH450 SEC, 2.5 µm Columns o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7um Columns Monoclonal Antibody Application o SEC-MS Applications Insulin and Small Protein Applications Factors Influencing Component Resolution Considerations to extending column life 2012 Waters Corporation 59
60 UP-SEC Recommendations H Class Bio system Biocompatible system for the analysis of biological molecules o Eliminate system corrosion o Best sample recovery Limit sample adsorption Limit damage to molecules, especially oxidation o Eliminate adduct formation in MS detection Based on ACQUITY UPLC H-Class System (Quaternary) True UPLC performance Compatible for all modes of chromatography Incorporates Auto Blend Plus Technology 2012 Waters Corporation 60
61 Excipients Added to increase protein stability, minimize protein-protein interactions Inhibit adsorption of proteins to vials Can affect protein aggregation Can affect biotherapeutic efficacy and immunogenicity Common excipients Carbohydrates (Sucrose, Treahlose) Surfactants (Triton X-100, Polysorbate (Tween) 80, Polysorbate (Tween) 20, Brij 35, Puronic F-68) Human serum albumin 2012 Waters Corporation 61
62 Column Lifetime USP Plate Count Column and guard Column without guard Injection Number Figure 1: Effect of using a 30 mm guard column on column efficiency for a monoclonal antibody. The arrows indicate where the guard column was changed Waters Corporation 62
63 Effect of Column Guard on Lifetime : Monoclonal Antibody No Guards Guards Replaced every 200 injections Injection 6 Injection Injection 2 Injection mab formulation with excipients (Tween 80) Improved mab peak tailing with use of guard column 2012 Waters Corporation 63
64 BEH200 SEC, 1.7um Care and Use: (Ways to extend column life) Preparation of SEC Mobile Phase and Needle Wash Pre filter through <0.2 um filter (i.e, Don t inject particulates) Use high purity water Replace mobile phases weekly and do not top off Ramp up and down flow to column over 1min to minimize bed shock Attention to SEC Eluent Inlet Filters Use titanium, NOT stainless steel Inlet filters can be major source of bacterial contamination o Consider occasional sinker replacement or 70% alcohol pull through to prevent problems Column Storage Considerations - Overnight: Continuously flush with the mobile phase at 10% of the maximum recommended flow rate - Extended: Store in the HPLC grade water with 10% methanol 2012 Waters Corporation 64
65 Bacterial growth Injection 627 Injection Injection Injection System pressure increases slightly over lifetime (~50 psi) Analysis of frit indicated bacterial growth 2012 Waters Corporation 65
66 Interaction with Flow Cell under Native Conditions Titanium 5 mm flow cell Standard Teflon AF 10mm flow cell SEC-PDA chromatogram of bovine serum album (BSA) (5 mg/ml in H20) shows the effect of flow cell material on peak shape. BSA monomer exhibits extensive peak tailing. Conditions: 25mM sodium phosphate, 150mM sodium chloride, ph 6.8, 0.4 ml/min, Injection volume: 4 µl, Wavelength: 280 nm; Column: ACQUITY UPLC BEH200 SEC 1.7 µm column, 4.6 x 300mm 2012 Waters Corporation 66
67 Auto Blend Plus Technology AQUITY H-Class Bio Program methods directly in units of ph and Molarity Calculation of required proportions from physical constants ph is calculated using Henderson-Hasselbalch equation with pka provided Typically use pka corrected for salt concentration OR: Empirical calibration table covering operating range of buffer and salts selected Proportions calculated at each pump stroke for best fidelity Independent gradients for ph and salt concentration Can produce a near true linear ph gradients as required 2012 Waters Corporation 67
68 Instrument Control Method Auto Blend to Auto Blend Plus Click to Convert to Auto Blend Plus TM 2012 Waters Corporation 68
69 Typical Gradient Table Auto Blend Plus 2012 Waters Corporation 69
70 Auto Blend Plus PH and Salt Gradients 2012 Waters Corporation 70
71 Calibration Options Auto Blend Plus Select to Use Empirical rather than Henderson- Hasselbalch 2012 Waters Corporation 71
72 Auto Blend Plus Calibration Options - Delivered ph Measured ph Programmed pk Corrected pk Empirical Fraction Number 2012 Waters Corporation 72
73 Auto Blend Plus Method Development ph 6.1 A B C ph 6.9 A B C 0.10 ph 7.0 A+B C ph 7.1 B A C ph 7.6 B A C A mixture of proteins was separated using cation exchange chromatography- alpha- Chymotrypsinogen A (peak A), Ribonuclease A (peak B), and Cytochrome C (peak C) 2012 Waters Corporation 73
74 SEC of Humanized Monoclonal Ab Effect of AutoBlend Plus ph Adjustment A: 17.26% B: 2.74% C: 2% D: 6% 125mM NaH 2 PO 4 125mM Na 2 H 2 PO mM NaCL H 2 O ph A: 2.74% B: 17.26% C: 2% D: 6% ph Waters Corporation 74
75 Developing a Robust SEC Method using AutoBlend Plus [NaCl] ph Monomer HMW 150 mm 250 mm 350 mm LMW 1 LMW UV 280nm Waters Corporation 75
76 Reference Material Care and Use Size Exclusion and Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC System, , REV. A Size Exclusion and Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC H-Class System, , Rev A Controlling contamination in LC/MS and HPLC/MS Systems, Improving the Lifetime of UPLC Size-Exclusion Chromatography Columns Using Short Guard Columns, Waters Technical Brief, en Guidelines for Routine Use and Maintenance of Ultra-Performance Size-Exclusion and Ion-Exchange Chromatography Systems, Waters Technical Brief, en 2012 Waters Corporation 76
77 Summary: Waters ACQUITY UPLC SEC System Solution New SEC column chemistries in 125Å, 200Å and 450Å pore sizes based on BEH particles Reduced secondary interaction Improved physical and chemical column lifetime Improved column-to-column reproducibility Improved resolution Improved throughput UPLC-SEC provides improved resolution, sensitivity, and higher throughput as compared to tradition HPLC Improved resolution of monoclonal antibody aggregates and clipped forms Complete system solution includes column chemistry and system UPLC columns specifically designed for bioseparations ACQUITY H-Class Bio System designed for the bioapplications Auto Blend Plus Technology provides convenience and efficiency 2012 Waters Corporation 77
78 Acknowledgements Paula Hong Kenneth Fountain Ed Bouvier Sue Serpa Bill Warren 2012 Waters Corporation 78
2012 Waters Corporation 1
UPLC User meeeting April 2012 Principles and Practices for SEC, IEX for Intact Protein Analysis by UPLC anders_feldthus@waters.com 2012 Waters Corporation 1 Agenda Ion-Exchange Chromatography Theory and
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