Analysis of biomolecules by SEC and Ion-Exchange UPLC

Transcription

1 Analysis of biomolecules by SEC and Ion-Exchange UPLC Anders Feldthus, Waters Nordic 2011 Waters Corporation 1

2 Waters Commitment To develop, commercialize and market columns that when used on Waters ACQUITY UPLC systems, give the speed, sensitivity, resolution, and method reproducibility that has not been previously achieved for the characterization of biological macromolecules with traditional HPLC Waters Corporation 2

3 Agenda Ion-Exchange Chromatography Theory and practice Protein-Pak Hi Res IEX Columns Method Development Strategies o Auto Blend Plus Technology and ACQUITY UPLC H-Class Bio System Size-Exclusion Chromatography Theory and practice ACQUITY UPLC for SEC o ACQUITY BEH200 SEC, 1.7 µm Columns Factors Influencing Component Resolution Ways to Maximize SEC Column Life 2011 Waters Corporation 3

4 Ion-Exchange Chromatography Cl - Na + Cl - Na + Cl - Na + Na + Cl - Anion- Exchange Particle Anion- Exchange Particle Cl - Na + Cl - Na + Cl - Cl - Cl - Na + Cl - Na + Cl - Na + 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) 2011 Waters Corporation 4

5 Ion-Exchange Gradient: Starting gpoint Sample High Equilibration Injection Gradient Salt Re-Equilibration Volume Wash unbound proteins ~ 5 cv cv 5-10 cv 5-10 cv Typical linear gradient for ion-exchange chromatography 2011 Waters Corporation 5

6 Ion-Exchange Advantages Moderate resolution Concentrating technique Limitations Separation by charge and charge distribution; insensitive to other properties Can load large volumes of a dilute sample For salt gradients, timeconsuming ph optimization Non-denaturing protein elution techniques Preferred first step when a cost-effective affinity purification is not available required for best resolution. Fractions may need to be desalted prior to the next purification or characterization step (e.g., Mass Spectrometry) 2011 Waters Corporation 6

7 Protein Isoelectric Points and IEX Particle Support Particle Support 2011 Waters Corporation 7

8 Strong versus Weak Ion Exchangers Strong and weak DO NOT indicate how tightly the protein is bound A strong exchanger is always ionized i while a weak exchanger s ionization varies with ph Weak Cation Exchange column- pk a Weak Anion Exchange column pk a ~9 Strong exchangers are recommended where there is a need to run at extreme ph (wider operating range) The ion exchange capacity of a weak ion exchanger varies with ph Sample loading (binding) capacity can vary with ph due to loss of charge from the exchanger Selectivity differences exist between weak and strong exchangers Binding constants are a function of the IEX functional group 2011 Waters Corporation 8

9 Agenda Ion-Exchange Chromatography Theory and practice Protein-Pak Hi Res IEX Columns Method Development Strategies o Auto Blend Plus Technology and ACQUITY UPLC H-Class Bio System Size-Exclusion Chromatography Theory and practice ACQUITY UPLC for SEC o ACQUITY BEH200 SEC, 1.7 µm Columns Factors Influencing Component Resolution 2011 Waters Corporation 9

10 Protein-Pak Hi Res IEX 2011 Waters Corporation 10

11 Attributes of Protein-Pak Hi Res IEX Columns Multi-layered network of ion-exchange groups (SP, CM or Q) o Effective diffusion and binding o High sample loading and resolution o 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 d pressures (up to 1450 psi for CEX and 2175 psi for AEX) Can be cleaned with aggressive washing ecord enabled for data tracking 2011 Waters Corporation 11

12 Batch to Batch Reproducibility: Protein-Pak Hi Res Q Column Retention Time Std Dev %RSD 1 - Myoglobin apo-transferrrin Trypsin Inhibitor Batch A Batch B Batch C Reproducible retention time observed for multiple batches 2011 Waters Corporation 12

13 Carryover st Repeat Gradient 2 nd Repeat Gradient Expected Elution Points No detectable carryover of lysozyme (100 µg injection) 2011 Waters Corporation 13

14 Ion-Exchange Analysis of Antibodies on Weak Cation Exchange Column Humanized IgG Humanized IgG Chimeric IgG Column: Protein-Pak Hi Res CM, 4.6 x 100mm 2011 Waters Corporation 14

15 Confirmation of c-terminal Lysine Variants of mab Untreated mab K KK mab treated with carboxypeptidase B, 30 C, 20 minutes IEX can be used to confirm the presence of mab lysine variants Column: Protein-Pak Hi Res CM, 4.6 x 100mm 2011 Waters Corporation 15

16 Agenda Ion-Exchange Chromatography Theory and practice Protein-Pak Hi Res IEX Columns Method Development Strategies o Auto Blend Plus Technology and ACQUITY UPLC H-Class Bio System Size-Exclusion Chromatography Theory and practice ACQUITY UPLC for SEC o ACQUITY BEH200 SEC, 1.7 µm Columns Factors Influencing Component Resolution Ways to Maximize SEC Column Life 2011 Waters Corporation 16

17 Strategies to Developing an Ion-Exchange Protein Separation Retention is optimized by adjustment of ionic strength Selectivity is most conveniently optimized with ph Changing buffer and counter ion may improve selectivity Methods may require adjustment if the temperature is changed 2011 Waters Corporation 17

18 Affect of Salt Gradient Slope % Elutes in high salt wash step M NaCl 3 5 Ovalbumin 1 Myoglobin 2 4 Ribonuclease A M NaCl 4 5 Cytochrome C 4 Lysozyme 5 % Unbound proteins % M NaCl Higher salt gradients result in earlier elution of bound proteins High salt wash may be needed in shallower gradients to elute tightly bound proteins Column: Protein-Pak Hi Res CM 4.6 mm x 100mm 2011 Waters Corporation 18

19 Affect of Salt Gradient Slope: Ovalbumin Variants 8 10 cv gradient cv gradient cv gradient Longer gradient: increased resolution, lower sensitivity Column: Protein-Pak HI Res Q, 4.6 x 100 mm 2011 Waters Corporation 19

20 Strategies to Developing an Ion-Exchange Protein Separation Retention is optimized by adjustment of ionic strength Selectivity is most conveniently optimized with ph Changing buffer and counter ion may improve selectivity Methods may require adjustment if the temperature is changed 2011 Waters Corporation 20

21 Effect of ph on Selectivity ph α-chymotrypsinogen 1 Ribonuclease A cytochrome c ph Column: Protein-Pak Hi Res CM 4.6 x 100 mm column 2011 Waters Corporation 21

22 Strategies to Developing an Ion-Exchange Protein Separation Retention is optimized by adjustment of ionic strength Selectivity is most conveniently optimized with ph Changing buffer and counter ion may improve selectivity Methods may require adjustment if the temperature is changed 2011 Waters Corporation 22

23 Effect of Buffer on Selectivity mM Sodium Phosphate 3 α-chymotrypsinogen Ribonuclease A cytochrome c mM MES Morpholino ethanesulfonic acid Buffer can alter selectivity and retention of proteins at same ph (6) Column: Protein-Pak Hi Res CM 4.6 x 100 mm column 2011 Waters Corporation 23

24 Counter Ion Effects Ribonuclease A 1 cytochrome c 2 Aprotinin NaCl KCl Counter ion may change selectivity it and retention ti of proteins Effects tend to be minimal 2011 Waters Corporation 24

25 Strategies to Developing an Ion-Exchange Protein Separation Retention is optimized by adjustment of ionic strength Selectivity is most conveniently optimized with ph Changing buffer and counter ion may improve selectivity Methods may require adjustment if the temperature is changed 2011 Waters Corporation 25

26 Temperature Effects C C C KK K 30 C Temperature may effect selectivity and retention Changes may be similar to those observed with ph change Column: ProteinP-ak Hi Res CM 4.6 x 100 mm column 2011 Waters Corporation 26

27 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 27

28 Auto Blend Plus Technology Enter Buffer Concentration Enter Buffer System Enter ph and Salt Gradient 2011 Waters Corporation 28

29 Effect of ph on Selectivity ph Ribonuclease A 1 cytochrome c 2 Aprotinin ph ph ph ph ph can be used to alter selectivity of proteins Column: Protein-Pak Hi Res CM 4.6 x 100 mm column 2011 Waters Corporation 29

30 AutoBlend Plus Buffer Management for Protein Ion Exchange Method Development Fluid Path of ACQUITY UPLC H-Class Bio System configured for multi-buffer use The optional solvent select valve provides 6 selectable choices that are associated with particular method names. The proportions of the four selected reservoirs are calculated with Auto Blend Plus Technology to meet the requirements of the method, using the defined buffer system specified with the method. The four physical reservoirs, labeled A, B, C, and D1-D6 can be assigned as needed as the Acid, Base, Salt, and Aqueous components Waters Corporation 30

31 Enhanced Method Development via AutoBlend Plus and Automated Column Management Automated Column Management for Protein Ion Exchange. The method specifies the column to be used and the bypass (B) permits matching the buffer to the column Waters Corporation 31

32 Use of AutoBlend Plus and Automated Column Switching for mab Separation Method Scouting Protein-Pak i k HiRes Q Protein-Pak HiRes SP Waters Corporation 32

33 IEX Summary Total system solution with combination of ACQUITY UPLC H Class Bio System and column Auto Blend Plus Technology allows for change of ph and ionic strengths without preparation of different buffers Simplifies methods development Protein-Pak Hi Res IEX column benefits Consistent batch-to-batch performance (tested with protein standards) Minimal column related carryover Stable over a wide ph range 2011 Waters Corporation 33

34 Agenda Ion-Exchange Chromatography Theory and practice Protein-Pak Hi Res IEX Columns Method Development Strategies o Auto Blend Plus Technology and ACQUITY UPLC H-Class Bio System Size-Exclusion Chromatography Theory and practice ACQUITY UPLC for SEC o ACQUITY BEH200 SEC, 1.7 µm Columns Factors Influencing Component Resolution Ways to Maximize SEC Column Life 2011 Waters Corporation 34

35 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 2011 Waters Corporation 35

36 Size Exclusion Separation of Proteins 2011 Waters Corporation 36

37 Common Customer Concerns Column-to-column reproducibility Changes in retention time Changes in spacing between peaks Changes in resolution Column lifetime Peak shape deteriorates over time Increased pressure Changes in resolution Tailing of specific proteins Resolution Throughput 2011 Waters Corporation 37

38 Agenda Ion-Exchange Chromatography Theory and practice Protein-Pak Hi Res IEX Columns Method Development Strategies o Auto Blend Plus Technology and ACQUITY UPLC H-Class Bio System Size-Exclusion Chromatography Theory and practice ACQUITY UPLC for SEC o ACQUITY BEH200 SEC, 1.7 µm Columns Factors Influencing Component Resolution Ways to Maximize SEC Column Life 2011 Waters Corporation 38

39 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 2011 Waters Corporation 39

40 Effect of LC System Dispersion on BEH200 SEC mab Separation Larger system dispersion decreases component resolution USP Res= 1.37 HPLC System BEH200 SEC 1.7um Column (4.6 x 300mm) Waters ACQUITY UPLC System 0.20 USP Res= 2.37 BEH200 SEC 1.7um 0.15 Column (4.6 x 300mm) Waters Corporation 40

41 UPLC-SEC vs HPLC-SEC of mab monomer and aggregates g ACQUITY BEH200 SEC, 1.7 µm 4.6 x 300mm HPLC 100% Silica-Diol SEC 250Å 5µm 7.8 x 300 mm % Aggregate % Aggregate Waters Corporation 41

42 ACQUITY BEH200 SEC 1.7 µm Columns Application Areas Determination of protein molecular weight Molecular weight range of 10,000 to 450,000 Daltons Determination of size heterogeneity in a protein sample Quantitation of protein aggregates primarily in therapeutic monoclonal antibodies Waters Corporation 42

43 BEH Overview The packing material is based on our patented Bridged Ethyl Hybrid base particle and effective diol bonding, which h provide a stable chemistry with minimal secondary interactions Waters Corporation 43

44 BEH200 SEC, 1.7um Column Batch Test Analyte pi MW Thyroglobulin, 3 mg/ml , IgG, 2 mg/ml (Vicam) , BSA, 5 mg/ml , Myoglobin, 2 mg/ml 6.8, , Uracil, 0.1 mg/ml N/A Waters Corporation 44

45 BEH200 SEC, 1.7um Calibration Curve MW IgG (~150,000 Da) Analyte MW Blue Dextran 2,000,000 Thyroglobulin 669,000 β-amylase 200, IgG, 150,000 Amyloglucosidase Conalbumin BSA 66,400 Ovalbumin 44,000 Carbonic Anhydrase 29,000 Myoglobin 17,000 Lysozyme 14,400 Ribonuclease A 13,700 Aprotinin 6,500 Uracil Elution Volume 2011 Waters Corporation 45

46 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 2011 Waters Corporation 46

47 DIOL coating used to minimize non-desired ionic interactions between proteins and packing material Ionic interactions between available negatively charged silanols on SEC particles and positive charges on proteins SEC Particle Waters Corporation 47

48 DIOL Loss increases non-desired ionic interactions between proteins and packing material SEC Particle Waters Corporation 48

49 Comparative SEC Column Life 0.70 Lysozyme, 0.60 pki = 10.7 HPLC 100% Silica-Diol 0.50 SEC 250Å 4µm 4.6 x 300 mm 0.40 Suggestive of DIOL Bleed 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 2011 Waters Corporation 49

50 NaCl will decrease ionic interactions between proteins and packing material Ionic interactions between available negatively charged silanols on SEC particles and positive charges on proteins Na + Cl - Cl - Na + Cl - Na + Cl - SEC Particle Na + Na + Na + Na + Cl - - Na + Na + Cl - Na + Cl - Cl - Cl - Cl - Na Waters Corporation 50

51 Influence of Ionic Strength on Peak Shape and Retention on Silica-based SEC Conventional 100% Silica-Diol Coated SEC Column 4.6 x 300 mm Lysozyme, pki = mM NaCl lysozyme 25mM NaCl lysozyme 100mM NaCl Flow rate: 0.5 ml/min; Mobile phase: 10, 25 or 100 mm sodium phosphate, ph Waters Corporation 51

52 Influence of Ionic Strength on Peak Shape and Retention on BEH200 SEC 0.24 ACQUITY BEH200 SEC 1.7 µm Column, x 150mm Lysozyme, 10mM NaCl 0.06 pki = mM NaCl mM 0.12 NaCl Flow rate: 0.5 ml/min; Mobile phase: 10, 25 or 100 mm sodium phosphate, ph Waters Corporation 52

53 Agenda Ion-Exchange Chromatography Theory and practice Protein-Pak Hi Res IEX Columns Method Development Strategies o Auto Blend Plus Technology and ACQUITY UPLC H-Class Bio System Size-Exclusion Chromatography Theory and practice ACQUITY UPLC for SEC o ACQUITY BEH200 SEC, 1.7 µm Columns Factors Influencing Component Resolution Ways to Maximize SEC Column Life 2011 Waters Corporation 53

54 Factors Influencing Resolution Resolution decreases with increasing volume and mass loads Resolution decreases with increasing flow rate Ideal flow rate is lower than typically running, however run time will be longer Resolution increases with column length Baseline resolution typically achieved at 50%-100% molecular weight difference Proteins with less than 100% molecular weight difference may not be acceptably resolved 2011 Waters Corporation 54

55 Mass Loading Capacity on BEH200 SEC, 1.7um 4.6 x 150mm µg Total Load Rs = µg Total Load Rs = 1.5 BSA BSA BSA dimer BSA dimer Constant injection volume, 20 µl Resolution decreases for mass loads of µg 2011 Waters Corporation 55

56 Volume Load Capacity on BEH200 SEC, 1.7um 4.6 x 150mm Injection Volume USP Resolution (Monomer-Dimer) 20 µl µl Constant BSA concentration, 20 mg/ml Resolution decreases for higher injection volume Poor peak shape observed at higher volumes 2011 Waters Corporation 56

57 Effect of Column Length on mab Rs 20.1% BEH200, SEC, 1.7um Aggregate 4.6 x 150 mm USP Res= % 9% Aggregate BEH200, SEC, 1.7um 4.6 x 300 mm USP Res= Comparable aggregate quantitation Human IgG (Sigma), (load: 10 µg - 150mm; 20 µg -300mm): TUV: 280 nm 2011 Waters Corporation 57

58 Effect of Flow Rate on mab Rs ml/min BEH200, SEC, 1.7um 4.6 x 150 mm ml/min ml/min Triplicate injections No observable trend in aggregation with flow rate 2011 Waters Corporation 58

59 Effect of Temperature on mab SEC Peak Shape C 45 C BEH200, SEC, 1.7um 4.6 x 150 mm C Chimeric IgG 0.4 ml/min, 25 mm Sodium Phosphate, 0.15m NaCl, ph Waters Corporation 59

60 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 2011 Waters Corporation 60

61 Agenda Ion-Exchange Chromatography Theory and practice Protein-Pak Hi Res IEX Columns Method Development Strategies o Auto Blend Plus Technology and ACQUITY UPLC H-Class Bio System Size-Exclusion Chromatography Theory and practice ACQUITY UPLC for SEC o ACQUITY BEH200 SEC, 1.7 µm Columns Factors Influencing Component Resolution Ways to Maximize SEC Column Life 2011 Waters Corporation 61

62 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 or 0.45 um filter (i.e, Don t inject particulates) Use high purity water Replace mobile phases weekly and do not top off Use of BEH200 SEC, 1.7um Guard Column Attention to SEC Eluent Sinkers Use titanium sinkers NOT stainless steel Sinkers 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 10-20% of the maximum recommended flow rate - Extended: Store in the HPLC grade water with 20% methanol 2011 Waters Corporation 62

63 BEH200 SEC, 1.7um Guard E(4.6 x 30mm) Extends UPLC SEC Column Life 2011 Waters Corporation 63

64 QC Protein Standards Mix on Bacterial Contaminated, BEH200 SEC Column BEH200, SEC, 1.7um 4.6 x 150 mm 2011 Waters Corporation 64

65 Summary: Waters ACQUITY UPLC SEC System Solution New SEC column chemistry based on BEH particles True UPLC separation Application benefits Reduced secondary interaction Improved physical and chemical column lifetime Improved column-to-column reproducibility Improved resolution Improved throughput Synergistic combination of UPLC system and column Higher throughput compared to traditional HPLC 2011 Waters Corporation 65