2012 Waters Corporation 1

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1 UPLC User meeeting April 2012 Principles and Practices for SEC, IEX for Intact Protein Analysis by UPLC 2012 Waters Corporation 1

2 Agenda Ion-Exchange Chromatography Theory and practice Protein-Pak P Hi Res IEX Columns Method Development Strategies Size-Exclusion Chromatography ACQUITY UPLC for SEC o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7 µm Columns Insulin Analysis Combination of SEC and MS 2012 Waters Corporation 2

3 Ion-Exchange Chromatography Binds at Low Ionic Strength Elute with Step or Continuous 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) 2012 Waters Corporation 3

4 Select buffer ph Isoelectric Point of a Protein (pi) Isoelectric point (pi) Zero net charge at this ph 2012 Waters Corporation 4

5 Select buffer ph Isoelectric Point of a Protein (pi) ph below pi Protein has net +ve charge ph region cation exchange ph above pi Protein has net -ve charge ph range for anion exchange 2012 Waters Corporation 5

6 Ion Exchange Chromatography Buffers Select buffer with pka near to desired ph Buffer ions should have same charge as functional groups on packing material (PO 4- + for cation, Tris for anion) 2012 Waters Corporation 6

7 Common Customer Concerns Reproducibility between columns Not getting required resolution from the start Recovery and carryover 2012 Waters Corporation 7

8 Protein-Pak Pak Hi Res IEX 2012 Waters Corporation 8

9 Attributes of Protein-Pak 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 d pressures (up to 1450 psi for CEX and 2175 psi for AEX) Can be cleaned with aggressive washing ecord enabled for data tracking 2012 Waters Corporation 9

10 Strategies to Developing an Ion-Exchange Protein Separation Selectivity is most conveniently optimized with ph Retention is optimized by adjustment of ionic strength Changing buffer and counter ion may improve selectivity Methods may require adjustment if the temperature is changed 2012 Waters Corporation 10

11 Effect of ph on Selectivity ph α-chymotrypsinogen 1 Ribonuclease A cytochrome c ph Minutes Column: Protein-Pak Hi Res CM 4.6 x 100 mm column 2012 Waters Corporation 11

12 Fine tune ph ph Effect on mab Separation ph ph ph Minutes Column: Protein-Pak Hi Res CM 4.6 x 100 mm column Gradient: M NaCl, 20mM Sodium Phosphate in 40 min Flow: 0.5 ml/min 2012 Waters Corporation 12

13 Effect of Salt Gradient Slope % Elutes in high salt wash step M NaCl Ovalbumin 1 Myoglobin 2 Ribonuclease A Minutes 0-0.3M NaCl 4 5 Cytochrome C 4 Lysozyme 5 % Unbound proteins Minutes % M NaCl Minutes 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 2012 Waters Corporation 13

14 Effect of Salt Gradient Slope: Ovalbumin Variants 8 10 cv gradient cv gradient cv gradient Minutes Longer gradient: increased resolution, lower sensitivity Column: Protein-Pak HI Res Q, 4.6 x 100 mm 2012 Waters Corporation 14

15 Effect of Buffer on Selectivity mM Sodium Phosphate α-chymotrypsinogen 1 Ribonuclease A cytochrome c mM MES (Morpholino ethane 1 Sulfonic acid) Minutes Buffer can alter selectivity and retention of proteins at same ph (6) Column: Protein-Pak Hi Res CM 4.6 x 100 mm column 2012 Waters Corporation 15

16 Counter Ion Effects Ribonuclease A 1 cytochrome c 2 Aprotinin NaCl KCl Minutes Counter ion may change selectivity it and retention ti of proteins Effects tend to be minimal 2012 Waters Corporation 16

17 Temperature Effects C C C K KK 30 C Temperature may effect selectivity and retention Minutes Changes may be similar to those observed with ph change Column: ProteinP-ak Hi Res CM 4.6 x 100 mm column 2012 Waters Corporation 17

18 IEX Summary 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 Method Parameters to optimize are Selectivity it is most conveniently optimized i with ph Retention is optimized by adjustment of ionic strength Changing buffer and counter ion may improve selectivity Methods may require adjustment if the temperature is changed 2012 Waters Corporation 18

19 Agenda Ion-Exchange Chromatography Theory and practice Protein-Pak P Hi Res IEX Columns Method Development Strategies Size-Exclusion Chromatography ACQUITY UPLC for SEC o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7 µm Columns Insulin Analysis Combination of SEC and MS 2012 Waters Corporation 19

20 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 2012 Waters Corporation 20

21 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 Minutes Minutes Waters Corporation 21

22 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) Minutes 2012 Waters Corporation 22

23 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 23

24 BEH 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 24

25 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 112 U A Minutes 2012 Waters Corporation 25

26 BEH200 SEC, 1.7um Batch-to-Batch Reproducibility 0.05 Batch 1, Column Batch 1, Column Batch 1, Column Batch 2, Column Batch 2, Column Minutes 2012 Waters Corporation 26

27 What s new? BEH 125 SEC UPLC Column 15 cm/30 cm/guard Launched on January 10th Linear range from to Dalton Reference Description ACQUITY UPLC BEH125 SEC 1.7µm 4.6x30 Gd ACQUITY UPLC BEH125 SEC 1.7µm 4.6x150mm ACQUITY UPLC BEH125 SEC 1.7µm 4.6x300mm 2012 Waters Corporation 27

28 Calibration Curves for ACQUITY UPLC BEH200 and BEH125, SEC, 1.7 μm Columns Thyroglobulin (~ 669,000 Da) IgG (~ 150,000 Da) BEH200, SEC, 1.7um Conalbumin (~ 75,000 Da) Rnase A (~ 13,700 Da) Ovalbumin dimer (~ 88,000 Da) Ovalbumin (~ 44,000 Da) Aprotinin (~ 6,500 Da) Angiotensin II (~ 1,045 Da) BEH125, SEC, 1.7um Uracil (~ 112 Da) 2012 Waters Corporation 28

29 Resolution of Proteins and Peptides 1.50 ACQUITY UPLC BEH125 SEC 1.7um 4.6 x 300mm A Minutes BioSuite125 UHR SEC 4.6 x 300mm Minutes 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 29

30 Insulin Analyses by Traditional HPLC-SEC vs UPLC-SEC Waters Alliance HPLC System Insulin HMWP SEC 10 μm (7.8 x 300mm) Waters ACQUITY UPLC System BEH125, SEC, 1.7 μm (4.6 x 300mm) 2012 Waters Corporation 30

31 Column Stability for Insulin Analysis Retention Time USP Resolution ime n) Retention T (min USP Resolu ution 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 31

32 What about SEC MS? 2012 Waters Corporation 32

33 SEC-MS Humanized Monoclonal Antibody 1.8e : Diode Array Da Range: 6.757e-1 1.6e-2 1.4e e e 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 on BEH cm Post UV detection additive: ACN, 0.8% Formic acid Scan 2012 Waters Corporation 33

34 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_p pt8fa_ 2_ (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 34

35 Summary: Waters ACQUITY UPLC SEC System Solution New SEC column chemistries 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 2012 Waters Corporation 35

36 Multi-Mode Mode Chromatography Automated with ACQUITY UPLC H-Class Bio Mouse Ascites Fluid SEC ACQUITY ACQUITY UPLC BEH x150mm 0.5mL/min 20mM Na phosphate, ph mM NaCl Minutes Cation Exchange Protein-Pak Hi Res SP 4.6x100mm 0.5mL/min 20mM phosphate ph mM NaCl 20mins Anion Exchange Protein-Pak Hi Q 4.6x100mm 0.5mL/min 20mM Tris ph mM NaCl 20mins Minutes Minutes 2012 Waters Corporation 36

37 Interesting Application Notes/posters on IEX and SEC IEX Method Development of a Monoclonal Antibody and Its Charge Variants en on Method Development for Size-Exclusion Chromatography of Monoclonal Antibodies and Higher Order Aggregates en on Multi-Mode Analytical Separations of Proteins en on Improving the Lifetime of UPLC Size-Exclusion Chromatography Columns Using Short Guard Columns en on Technology Brief with SEC and IEX guidelines en on Technology Brief on SEC with MS en on Waters Corporation 37

38 Looking for more info? Waters Corporation 38