Simple Techniques for Improving the Isolation of Synthetic Peptides Jo-Ann Jablonski Principal Scientist Waters Corporation

Transcription

1 Simple Techniques for Improving the Isolation of Synthetic Peptides Jo-Ann Jablonski Principal Scientist Waters Corporation 2016 Waters Corporation 1

2 Agenda Background Techniques Scaling a separation Focusing the gradient At-column dilution Temperature control Practical example Summary 2016 Waters Corporation 2

3 Why peptides? Unique role Attractive as potential drug candidates Sources 2016 Waters Corporation 3

4 Synthetic Peptides Synthetic peptides Amino acids linked using organic chemical reactions Average: residues Large: residues or more Impurities Failure Sequences Deletion Sequences Damaged Sequences Cleavage Adducts Purification requirements 75-98% purity of final product 2016 Waters Corporation 4

5 Peptide Isolation Workflow Amount? Enough pure peptide? Yes No Pure peptide Crude analysis Isolation Fraction Analysis 2016 Waters Corporation 5

6 Most Common Operating Conditions Purification using reversed-phase chromatography C18 columns Elution with gradient Monitor with UV detection at nm Mass-directed isolation 2016 Waters Corporation 6

7 Agenda Background Techniques Scaling a separation Focusing the gradient At-column dilution Temperature control Practical example Summary 2016 Waters Corporation 7

8 Scaling Equations M M M L L d d Mass 2 L2 d 2 = M 1 where 2 L1 d 1 ismass oncolumn1 2 : ismass oncolumn2 islength of Column1 islength of Column2 isdiameterof Column1 isdiameterof Column2 t t t 1 2 L d d F F 2 L2 d 2 F 1 = t 1 where 2 L1 d 1 F 2 is Gradient Duration for Column1 2 : 2 is Gradient Duration for Column Gradient L1 islength of Column1 islength of Column2 isdiameterof Column1 isdiameterof Column2 isflow rate for column1 isflow rate for column2 F F F d d Flow rate 2 d 2 = F 1 where 2 d 1 isflow Rate for Column1 2 : isflow Rate Column2 is Diameter of Column1 isdiameterof Column Waters Corporation 8

9 Scaling a Method Analytical Method Time Flow %A %B Preparative Method Time Flow %A %B Column: 4.6 x 50 mm System dwell volume: 0.71 ml Column volume: ml System dwell volume (in column volumes): 1.02 Column: 19 x 50 mm System dwell volume: 4.75 ml Column volume: 11.9 ml System dwell volume (in column volumes): 0.40 Gradient offset: Waters Corporation 9

10 Gradient Scaling 100 % 4.6 x 50mm 1.46 ml/min 4 mg % 19 x 50 mm ml/min 68.2mg -1 Time Waters Corporation 10

11 Estimated Peptide Mass Loading Capacity (mg) Length (mm) Diameter (mm) * 19 * 30 ** * 5 µm and 10 µm OBD Prep Columns; ** 10 µm OBD Prep Columns * [OBD ], Optimum Bed Density; US Patent # 7,399,410, UK Patent # GB Estimates of mass load are broad because capacity depends on solubility of the peptide in the mobile phase Waters Corporation 11

12 Flow rate (ml/min) Injection Volume (µl) Diameter (mm) * 19 * 30 ** 50 Flow rate Inj Vol * 5 µm and 10 µm OBD Prep Columns; ** 10 µm OBD Prep Columns 2016 Waters Corporation 12

13 Agenda Background Techniques Scaling a separation Focusing the gradient At-column dilution Temperature control Practical example Summary 2016 Waters Corporation 13

14 Gradient Types Linear Segmented Focused Waters Corporation 14

15 Developing a Focused Gradient Estimate volume of system and column Make pilot run of sample From elution time, system volume and gradient table, estimate % that elutes the peptide From gradient and column volume, estimate gradient slope in pilot run in % change per column volume Create shallow gradient segment from 5% below to 3% above estimated elution percentage Full Description in Application Note: Developing Focused Gradients for Isolation and Purification en 2016 Waters Corporation 15

16 AU AU Crude Peptide Analysis nm nm Minutes XBridge Peptide BEH C 18 5µm Column, 4.6 x 100 mm Gradient: 5-50%B in 10 minutes, 3.4% change per column volume, 5 µl Peptide eluted at 33%B Waters Corporation 16

17 AU AU Focused Gradients %/cv 28-36%B in 9 minutes %/cv 28-36%B in 18 minutes Minutes 2016 Waters Corporation 17

18 Agenda Background Techniques Scaling a separation Focusing the gradient At-column dilution Temperature control Practical example Summary 2016 Waters Corporation 18

19 Dissolving Synthetic Peptides No universal technique or solvent Apparent insolubility may include synthesis by-products Useful choices Water with % TFA Wet with small amount of DMF, dilute with water to ~10% organic 100% DMSO 100% Hexafluoroisopropanol (HFIP) 6-12M Guanidine-HCl (not for use with mass spectrometers) Always results in relatively large volume of relatively strong solvent 2016 Waters Corporation 19

20 Standard Gradient System 2016 Waters Corporation 20

21 Standard Prep Separation With DMSO As Sample Solvent 2016 Waters Corporation 21

22 At-Column Dilution Waters Application Note : rA US Patent (Waters Corporation) 6,790,361 B Waters Corporation 22

23 At-Column Dilution Prep Separation 2016 Waters Corporation 23

24 Conventional vs. At-Column Dilution 2016 Waters Corporation 24

25 At-Column Dilution Permits injection of large volumes of strong solvents Improves both mass capacity and resolution Increases system ruggedness Extends column life Reduces introduction of particles Reduces pressure shock from viscous solvent Waters Application Note : rA US Patent (Waters Corporation) 6,790,361 B Waters Corporation 25

26 Agenda Background Techniques Scaling a separation Focusing the gradient At-column dilution Temperature control Practical example Summary 2016 Waters Corporation 26

27 AU AU Effect of Temperature Crude Synthetic Peptide 1.0e C e+1 1.2e+1 40 C 1.0e Time Waters Corporation 27

28 AU AU Effect of Temperature Peptide Separation 8.0e-3 7.0e e-3 5.0e-3 4.0e-3 3.0e-3 2.0e-3 1.0e e-3 7.0e e-3 5.0e-3 4.0e-3 3.0e-3 2.0e-3 1.0e-3 Time Waters Corporation 28

29 AU AU Effect of Temperature Peptide Separation 7.5e-3 7.0e-3 6.5e-3 6.0e e-3 5.0e-3 4.5e-3 4.0e-3 3.5e-3 3.0e-3 2.5e-3 2.0e-3 1.5e-3 1.0e e-3 6.0e-3 5.5e e-3 4.5e-3 4.0e-3 3.5e-3 3.0e-3 2.5e-3 2.0e-3 1.5e-3 1.0e-3 Time Waters Corporation 29

30 Controlling Temperature in Purification Practical Considerations Large diameter columns cannot be effectively heated from the outside High flow rate separations occur at the temperature of the incoming solvent Temperature gradient within column Add a coil of stainless steel tubing to the inlet end of the column for pre-heating (e.g. 5-mL sample loop) Immerse column and pre-heating coil into a water bath 2016 Waters Corporation 30

31 Column and Pre-heater Loop Immersed in Water Bath at Waters Corporation 31

32 At-Column-Dilution: Sample Loading with Temperature Control Sample in 100% DMSO carried to Tee by organic solvent Tee Initial 100% aqueous buffer generated by gradient pump Preheating loop 2016 Waters Corporation 32

33 Agenda Background Techniques Scaling a separation Focusing the gradient At-column dilution Temperature control Practical example Summary 2016 Waters Corporation 33

34 AU AU 3.23 min AU Temperature Evaluation Crude Peptide A 6.0e-1 4.0e-1 25 C 2.0e e-1 B 6.0e-1 4.0e-1 40 C 2.0e C 6.0e-1 4.0e-1 60 C 2.0e Time XBridge Peptide BEH C x 50 mm Gradient: 0-30% isopropanol in 5 40 C; 5.13 mg/ml, 7 µl 2016 Waters Corporation 34

35 Focused Gradient Screening Gradient Time Flow %A %B Focused Gradient Time Flow %A %B System volume: 0.77 ml Column volume: ml Calculated peptide elution %: 10.85% Screening gradient slope: 3.0%/column volume Focused gradient segment range: 5-13%B Calculated focused gradient slope: 0.6%/column volume Calculated time for the focused gradient segment: 6.37 min Gradient segment range: 5-13%B Gradient slope: 0.6%/column volume Time for the focused gradient segment: 6.37 min 2016 Waters Corporation 35

36 AU AU AU AU Loading Study Focused Gradient 3.0e-1 A 2.0e-1 7 µl, 36 µg 1.0e B 7.5e-1 5.0e-1 14 µl, 72 µg * 2.5e C e-1 28 µl, 144 µg D e-1 5.0e-1 50 µl, 256 µg 2.5e Time 5-13% isopropanol with 0.1% 40 C, 214 nm 2016 Waters Corporation 36

37 Scaling Analytical Method Time Flow %A %B Preparative Method Time Flow %A %B Column: 4.6 x 50 mm System dwell volume: 0.77 ml Column volume: ml System dwell volume (in column volumes): 1.10 Column: 19 x 100 mm System dwell volume: 9.75 ml Column volume: ml System dwell volume (in column volumes): 0.41 Gradient offset: Waters Corporation 37

38 AU Preparative Chromatography Conventional Injection 1.75e e e-2 1.0e-2 7.5e-3 5.0e-3 2.5e e-3-5.0e-3-7.5e-3-1.0e e-2-1.5e e-2-2.0e e-2-2.5e e-2-3.0e e-2-3.5e Time XBridge Peptide BEH C 18 5 µm OBD Prep, 19 x 100 mm, 2.4 mg 2016 Waters Corporation 38

39 AU AU AU Fraction Analysis A 2.0e-1 1.5e-1 1.0e-1 5.0e-2 Blank B 2.0e-1 1.5e-1 1.0e-1 5.0e-2 Fraction C 2.0e-1 1.5e-1 1.0e-1 5.0e-2 Fraction Time Waters Corporation 39

40 AU AU AU Fraction Analysis A 2.0e-1 1.5e-1 1.0e-1 5.0e-2 Blank without column e-1 Blank with column B 1.5e-1 1.0e-1 5.0e e-1 Fraction 9 C 1.5e-1 1.0e-1 5.0e Time Waters Corporation 40

41 Comparison of Prep Gradient Methods Conventional Injection Time Flow %A %B Gradient time: = min Gradient volume: min x 25 ml/min = ml Column volumes: ml x 1 cv/ ml = cv Gradient slope: 8%/13.38 cv = 0.6%/col vol At-Column Dilution Injection Time Flow %A %B Gradient time: = min Gradient volume: min x 25 ml/min = ml Column volumes: ml x 1 cv/ ml = cv Gradient slope: 8%/13.38 cv = 0.6%/col vol Loading pump flow rate during gradient = 1.25 ml/min 1.25 ml/min = 5% of the total flow Total gradient method flow rate = 25 ml/min 2016 Waters Corporation 41

42 At-Column Dilution with Temperature Control Sample in 100% DMSO carried to Tee by organic solvent Tee Initial 100% aqueous buffer generated by gradient pump Preheating loop 2016 Waters Corporation 42

43 AU AU Loading Method Comparison 8.0e-2 6.0e-2 Conventional 2.4 mg 4.0e-2 A 2.0e e-2 Time B 8.0e-2 7.0e-2 6.0e-2 5.0e-2 4.0e-2 3.0e-2 At-Column Dilution 12 mg 2.0e-2 1.0e-2 Time Waters Corporation 43

44 AU Preparative Chromatography At-Column Dilution 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 1.5e-2 1.0e-2 5.0e Time 2016 Waters Corporation 44

45 AU AU AU AU AU AU AU AU Fraction Analysis 3.0e-1 Blank 3.0e-1 2.0e-1 Fr e-1 1.0e-1 1.0e Fr e-1 Fr e-1 2.0e-1 2.0e-1 1.0e-1 1.0e Fr e-1 Fr e-1 2.0e-1 2.0e-1 1.0e-1 1.0e Fr e-1 Fr e-1 2.0e-1 2.0e-1 1.0e-1 1.0e Time Time Waters Corporation 45

46 Agenda Background Techniques Scaling a separation Focusing the gradient At-column dilution Temperature control Practical example Summary 2016 Waters Corporation 46

47 Summary Focusing the gradient improves resolution and load Temperature control is a useful tool for improving peptide detection, peak shape, and resolution At-Column Dilution permits injection of large volumes of strong solvents, improves mass capacity and resolution, increases system ruggedness, and extends column life 2016 Waters Corporation 47

48 Supplementary Information Developing Focused Gradients for Isolation and Purification Waters Technical Note, EN Effective Use of Temperature Control in Compound Isolation Waters Technical Note, EN At-Column Dilution Application Notes Waters Application Notes, Prep 150 LC System: Considerations for Analytical to Preparative Scaling Waters Application Note, EN Peptide Isolation Using the Prep 150 LC System Waters Application Note, EN Topics in Liquid Chromatography Part 2, Optimum Bed Density [OBD ] Columns Waters White Paper, EN 2016 Waters Corporation 48