Benefit of light scattering technologies (RALS/LALS/MALS) and multidetection characterization in life science research?

Transcription

1 Benefit of light scattering technologies (RALS/LALS/MALS) and multidetection characterization in life science research? Bert Postma Business Support Separations and MicroCal Size Exclusion Chromatography SEC has long been used as a key tool for measuring molecular weight or for purification SEC separates molecules according to their size on a specific chromatographic column Larger molecules elute first Molecules must be in solution Purely physical separation After the column, the separated molecules pass through one or more detectors Component A Component B Sample Solution 1

2 3 Malvern GPC/SEC light scattering LALS, RALS, MALS, DLS 2

3 OMNISEC An OMNISEC system is an out of the box solution for SEC Comprising a complete system for mobile phase and sample delivery and separation, and an integrated detector module for detailed molecular characterization Two parts OMNISEC RESOLVE OMNISEC REVEAL OMNISEC Separations module OMNISEC RESOLVE Sample characterization OMNISEC REVEAL 3

4 OMNISEC Separations module OMNISEC RESOLVE Detector Module OMNISEC REVEAL Low-volume (2 ml), high efficiency degasser Self-priming pump, all solvents 1 ul ul Inj.vol. Temperature controlled (4-60 C) vial holder Detector module OMNISEC REVEAL Redesigned LALS/RALS with significant sensitivity improvement New viscometer design (self-balancing bridge with easy-exchangeable capillaries) Improved RI, including change of position in detector module Completely new UV-diode array (full UV/Vis range) Temperature control of all detectors and inter-detector tubing (20 to 60 C) One single USB connection to PC 4

5 Triple Detection benefits LALS/RALS detector measures molecular weight and has the sensitivity for small Mw s and does measure directly large Mw. Viscometer offers additional measurements of: Hydrodynamic radius (1-200 nm) Mark-Houwink plots (a and K values), Structure analysis and comparison Integration benefits better chromatography and sensitivity reduced tubing length and reduced band broadening = better results (significantly improved in OMNISEC) OMNISEC software v10.0 5

6 OMNISEC software v10.0 OMNISEC is a completely new software platform Modern intuitive interface in line with latest consumer software packages Sequence setup 6

7 Data Acquisition Data analysis 7

8 Data overlays OMNISEC software v10 features Powerful analysis tab allows: Simultaneous analysis of multiple samples (from multiple sequences) at the same time Easily review and compare historical analyses of one or more chromatograms Simple customizable reports allow presentation of only the results that are most relevant to you Multi-chromatogram analysis gives true statistical analysis of repeat injections (Mean, standard deviation, RSD, Max, Min) Overlay all types of chromatograms and results 8

9 The benefits in moving from single to multi-detection in Size Exclusion Chromatography Contents Introduction to GPC/SEC Triple and Tetra detection Why use Multiple detection? Application Examples Conclusions 9

10 What type of molecules are we dealing with? Polymers Synthetic (polystyrene, polyurethanes, polyamides, etc ) Natural (polysaccharides such as cellulose, starches, pectins ) Artificial (modified natural polymers) Biological macromolecules Proteins Nucleic acids Other macromolecules Introduction Conventional SEC compares the retention volume of a sample with that of standards of known molecular weight using a single concentration detector Gives Relative Mw Concentration detector response Refractive Index Response (mv) 1472,0 36,0 1394,0 34,0 1312,0 32,0 1230,0 30,0 1148,0 28,0 1066,0 26,0 984,0 24,0 902,0 22,0 820,0 20,0 738,0 18,0 656,0 16,0 574,0 14,0 492,0 12,0 410,0 10,0 328,0 246,0 164,0 82,0 0,0 Viscometer DP Response (mv) 8,0 6,0 4,0 2,0 0,0? 5,30 5,6 5,8 6,0 6,2 6,4 6,6 6,8 7,0 7,2 7,4 7,6 7,8 8,0 8,2 8,4 8,6 8,8 9,0 9,2 9,4 9,610,00 Retention Volume (ml)?? 239,0 7, ,0 6, ,0 6, ,0 6, ,0 6, ,0 6, ,0 140,0 5,800 Log Molecular Weight 126,0 5, ,0 5,400 98,0 84,0 70,0 56,0 42,0 28,0 Right Angle Light Scattering Response (mv) 14,0 0,0 5,200 5,000 4,800 4,600 4,400 4,200 4,000 10

11 Traditional GFC to analyse proteins 21 Refractive Index Response (mv) 93,4 88,0 84,0 80,0 76,0 72,0 68,0 64,0 60,0 56,0 52,0 48,0 44,0 40,0 36,0 32,0 28,0 24,0 Concentration detector RI Calibration curve based on globular proteins A B Log Mw 20,0 16,0 12,0 8,0 4,0-0,8 11,7 12,0 12,3 12,6 12,9 13,2 13,5 13,8 14,1 14,4 14,7 15,0 15,3 15,6 15,9 16,2 16,5 16,8 17,1 17,4 17,7 18,0 18,3 RetentionVolume (ml) Effect of molecular shape on GPC/SEC retention volume Columns separate on size (Vh) not molecular weight Structural changes will affect results Retention volume 11

12 SEC Conventional SEC compares the retention volume of a sample with that of standards of known molecular weight using a single concentration detector Gives Relative Mw Advanced SEC adds specific detectors to make other measurements of the sample as it elutes light scattering viscometer The Triple detection pyramid Structure Indirectly calculate: Hydrodynamic radius (Rh) Mark-Houwink plot Directly calculate: Molecular weight Radius of gyration (Rg) Concentration + Composition Intrinsic viscosity Directly measure: Light scattering intensity δ Refractive Index UV absorbance δ viscosity 12

13 OMNISEC analysis: detector response Light Scattering = K LS. Molecular weight. (dn/dc) 2. Conc Refractometer = K RI. dn/dc. Concentration Viscometer = K V. Intrinsic Viscosity. Concentration Static Light Scattering (Mw) Light scattering can be used to measure the molecular weight of a macromolecule in solution A photon from an incident beam is absorbed by a macromolecule and re-emitted in all directions The intensity of the scattered light is proportional to the molecular weight of the macromolecule according to the Rayleigh equation. Molecular weight Intensity KC 1 2A2C R MWP 13

14 Intrinsic viscosity (IV) Intrinsic viscosity is how a dissolved molecule contributes to the overall viscosity of the solution Intrinsic viscosity is the inverse of molecular density Since structure is inextricably tied to density, IV is a measure of molecular structure IV lim sp c c 0 0 sp 0 sp is called the specific viscosity of the solution whose concentration is C. o is the Solvent Viscosity. is the Solution Viscosity. Refractive index detector (conc.) Measure sample concentration Measure dn/dc OMNISEC contains a robust RI cell placing it at the front of detector series Minimizes band broadening Wavelength matched with LS detector (640 nm) Enhanced sensitivity 14

15 SO WHY USE TRIPLE DETECTION AND TETRA DETECTION? APPLICATION EXAMPLES Identification of the peaks and oligomeric state Absolute molecular weight of each peak, not relying on globular reference proteins Concentration detector response Refractive Index Response (mv) 1472,0 36,0 1394,0 34,0 1312,0 32,0 1230,0 30,0 1148,0 28,0 1066,0 26,0 984,0 24,0 902,0 22,0 820,0 20,0 738,0 18,0 656,0 16,0 574,0 14,0 492,0 12,0 410,0 10,0 328,0 8,0 246,0 6,0 164,0 4,0 82,0 2,0 0,0 0,0 Viscometer DP Response (mv) 5,30 5,6 5,8 6,0 6,2 6,4 6,6 6,8 7,0 7,2 7,4 7,6 7,8 8,0 8,2 8,4 8,6 8,8 9,0 9,2 9,4 9,610,00 Retention Volume (ml) 239,0 7, ,0 6, ,0 6, ,0 6, ,0 6, ,0 6, ,0 140,0 5,800 Log Molecular Weight 126,0 5, ,0 5,400 98,0 84,0 70,0 56,0 42,0 28,0 Right Angle Light Scattering Response (mv) 14,0 0,0 5,200 5,000 4,800 4,600 4,400 4,200 4,000 15

16 Traditional GFC to analyse proteins Refractive Index Response (mv) 93,4 88,0 84,0 80,0 76,0 72,0 68,0 64,0 60,0 56,0 52,0 48,0 44,0 40,0 36,0 32,0 28,0 24,0 Concentration detector RI Calibration curve based on globular proteins A B Log Mw 20,0 16,0 12,0 8,0 4,0-0,8 11,7 12,0 12,3 12,6 12,9 13,2 13,5 13,8 14,1 14,4 14,7 15,0 15,3 15,6 15,9 16,2 16,5 16,8 17,1 17,4 17,7 18,0 18,3 RetentionVolume (ml) Accurate Molecular weight from light scattering RI LS Log MW vs RV 93,4 88,0 84,0 80,0 12,9 12,0 11,0 11,951 11,600 11,200 10,800 76,0 10,400 A B MW 24.5 kda 28.5 kda Refractive Index Response (mv) 72,0 68,0 64,0 60,0 56,0 52,0 48,0 44,0 40,0 36,0 Right Angle Light Scattering Response (mv) 10,0 9,0 8,0 7,0 6,0 5,0 10,000 9,600 9,200 8,800 8,400 8,000 7,600 7,200 Log Molecular Weight 32,0 6,800 28,0 24,0 20,0 16,0 4,0 3,0 A B 6,400 6,000 5,600 12,0 2,0 5,200 8,0 1,0 4,800 4,0 4,400-0,8 0,0 4,020 11,4111,7 12,0 12,3 12,6 12,9 13,2 13,5 13,8 14,1 14,4 14,7 15,0 15,3 15,6 15,9 16,2 16,5 16,8 17,1 17,4 17,7 18,0 18,3 18,78 Retention Volume (ml) Conclusion: Protein A has a non-globular shape and consequently elutes earlier. 16

17 Conventional calibration: IgG MW estimation of two IgG samples by conventional chromatography. Human IgG 179 kda Sheep IgG 186 kda 25 o C, 2xP3000, 1ml/min, PBS, 100µL injection Triple Detection: IgG Human IgG Sheep IgG: monomer dimer trimer other Mw (kda) % composition Pd IV Rh 5.1 monomer dimer other Mw (kda) % composition Pd IV Rh

18 Triple detection: IgG Mw of ~150 kda is more in line with expectations. The monomer peak of Human IgG is less polydisperse better resolution between monomer and dimer peaks. There are compositional differences that can be quantified. Human IgG is more aggregated i.e. lower yield monomer. The monomer Rh of sheep IgG is larger than that of Human IgG which rationalises the differences in observed retention times. Human IgG monomer dimer trimer other Mw (kda) % composition Pd IV Rh 5.1 monomer dimer other Mw (kda) % composition Pd IV Rh 5.25 Pepsin aggregation Pepsin monomer is 35 kda A very large aggregate peak is clearly visible A polydisperse lower molecular weight peak is also present Since pepsin is a digestive enzyme, the lower molecular weight peaks are likely to represent products of degradation Aggregates Monomer Degradation products Peak RV (ml) Mn (kda) Mw (kda) Mw / Mn Rg(w) (nm) 69.9 N/C N/C Wt Fr (Peak)

19 TRIPLE DETECTION WHAT IS THE VISCOMETER TELLING US MORE THAN LIGHT SCATTERING ALONE? Beta amylase from sweet potato Beta amylase has an MW of 223 Kda It elutes as two peaks The peak used for the conventional calibration is the one at 16 ml, which is assumed to be the monomer The earlier eluting peak is broader and would generally be assumed to be some aggregated material R v = 14.6mL R v = 16.0mL 19

20 Triple detector data: Same MW, but different structure Peak 1 Peak 2 RV Mw (kda) IV Rh (nm) Similar molecular weight Significant increase in IV Larger IV indicative of decrease in density Peak 1 has more open structure supported by increase in Rh Purity of DNA samples: what is the shoulder on the UV signal? Ultra Violet Response (mv) Log Intrinsic Viscosity Log Molecular Weight Retention Volume (ml) 20

21 DNA - Recognition of Different Structures DNA Structure Molecular Weight in 10 6 g/mol Intr. Viscosity in dl/g Hydrodyn. Radius in nm Closed Coil (CC) 3,2 4,6 62,8 Open Coil (OC) 3,2 9,1 77,9 Linear 3,2 12,2 86,8 Purity of DNA samples Ultra Violet Response (mv) IV =12.5 IV = Log Intrinsic Viscosity Log Molecular Weight IV of linear DNA matches IV of shoulder in Open Coil sample. So shoulder is contamination by linear DNA Retention Volume (ml) 21

22 A little more about the viscometer and how the viscometer gives the evidence Conformational Changes Adenylate Cyclase Toxin is an intrinsically disordered protein. Adopts active form in the presence of calcium ions. MW IV R h - Ca 78, Ca 77, fold 2-fold Conclusion: Significant differences in IV and size reflect significant structural changes upon calcium binding App. Note: MRK Paper: A. Chenal, J. Biol. Chem., 2009; 284: _13;55;40_Ca-holo-RD_01.vdt / Method: EDTA vcm QUESTION: Are the non-active and active conformations of Adenylate Cyclase Toxin different? _13;55;40_Ca-holo-RD_01.vdt / Method: EDTA vcm Retention Volume (ml) Retention Volume (ml) RI VISCOMETER 22

23 Tetra detection Why using 2 concentration detectors? Identifying PEGylated Proteins To determine if PEG-Protein conjugation has been successful we must identify each sample component within the chromatogram. Perform conjugate analysis RI LS UV Detector Response (mv) LS RI UV Data File: 006A RXN SOLN (6.vdt) Method: PEG-Prot-0002.vcm 9,91 10,82 11,73 12,64 13,55 14,46 15,37 16,28 17,19 18,10 19,01 Retention Volume (ml) 23

24 Identifying PEGylated Proteins Produce concentration profile of each material Identity and concentration of each component can be determined. Protein concentration 1,8 e-4 1,6 e-4 1,4 e-4 1,2 e-4 1,1 e-4 8,8 e-5 7,0 e-5 5,3 e-5 Protein-PEG Aggregates PEG Protein Monomer 2,1 e-4 1,9 e-4 1,7 e-4 1,5 e-4 1,3 e-4 1,1 e-4 8,5 e-5 6,3 e-5 PEG concentration 3,5 e-5 4,2 e-5 1,8 e-5 2,1 e-5 0,0 0,0 12,00 12,8 13,2 13,6 14,0 14,4 14,8 15,2 15,6 16,0 16,4 16,8 17,2 17,6 18,0 18,4 19,00 Retention Volume (ml) Conclusion: Protein and polymer do not co-elute. Conjugation has been unsuccessful. Conjugate Analysis The conjugate analysis uses two concentration detectors RI UV By comparing the two detectors responses we can determine the concentration of two components. Glycosylated proteins e.g. antibodies Protein-polymer complexes e.g. PEGylated proteins Membrane detergent complexes DNA encapsulated Liposomes Protein-DNA interaction 24

25 Identifying Conjugated Proteins After further method development, the components co-elute. 5,5 e-4 5,0 e-4 4,4 e-4 Protein-PEG conjugate 3,5 e-4 3,1 e-4 2,8 e-4 Protein concentration 3,9 e-4 3,3 e-4 2,8 e-4 2,2 e-4 1,7 e-4 1,1 e-4 5,5 e-5 Protein-PEG aggregates 2,4 e-4 2,1 e-4 1,7 e-4 1,4 e-4 1,0 e-4 7,0 e-5 3,5 e-5 PEG concentration 0,0 10,00 10,5 10,8 11,1 11,4 11,7 12,0 12,3 12,6 12,9 13,2 13,5 13,8 14,1 14,4 14,7 15,0 15,3 15,6 16,00 Retention Volume (ml) 0,0 Conclusion: Protein-polymer conjugation has been successful. Identifying PEGylation extent Conc (Protein+PEG) 9,0 e-4 8,1 e-4 7,2 e-4 6,3 e-4 5,4 e-4 4,5 e-4 3,6 e-4 2,7 e-4 1,8 e-4 9,0 e-5 0,0 Conc Protein 9,0 e-4 8,1 e-4 7,2 e-4 6,3 e-4 5,4 e-4 4,5 e-4 3,6 e-4 2,7 e-4 1,8 e-4 9,0 e-5 0,0 Conc PEG 9,0 e-4 8,1 e-4 7,2 e-4 6,3 e-4 5,4 e-4 4,5 e-4 3,6 e-4 2,7 e-4 1,8 e-4 9,0 e-5 0,0 Conc. Protein (18 kda) Conc. PEG (3 kda) Conc. Protein + PEG Higher order structure with PEG 1x Protein + 4x PEG 30 kda 9,90 10,5 10,8 11,1 11,4 11,7 12,0 12,3 12,6 12,9 13,2 13,5 13,8 14,1 14,4 14,7 15,0 15,3 15,80 Retention Volume (ml) 5,500 5,400 5,200 5,000 4,800 4,600 4,400 4,200 4,000 3,800 3,600 3,500 Log Molecular Weight 25

26 Polysaccharides and derivatives Natural polymers Natural polymers are derived from natural products and are most commonly different forms of polysaccharides They are commonly used in food and medicinal products Like synthetic polymers, the molecular properties of synthetic polymers relate to their bulk properties In food products, molecular weight and structure will affect mouth-feel In medicinals, molecular weight and structure affect viscosity and efficiency among other things Understanding these properties is again key to a robust process 26

27 Comparison of polysaccharides When the measurements of different polysaccharides are compared, the differences are clear but the meaning of those differences is less clear It is useful to compare them visually Sample Id Dextran Pectin Gum Arabic Pullulan HPC Injection Mn (kda) Mw (kda) Mz (kda) , IV (dl/g) Rh (nm) Rg (nm) Compare samples in more details Dextran Gum Arabic Pectin Pullulan Hydroxypropyl cellulose 27

28 Exploring effects of modification using Mark Houwink plots (MW and IV together) (a) Polymeric starting material (b) with substitution/ addition/ modification of side chains Viscometer for Conformational Analysis and Mark-Houwink Plot The analysis of how the intrinsic viscosity changes across the molecular weight distribution Provides a molecular density profile Density is related back to structure log Density KM w log K a log M a Structure w Increasing Density a < 0.5 Compact/spherical chains 0.5 < a < 0.8 Random-coil/flexible chains a > 0.8 rigid-rod/stiff chains 28

29 Polysaccharide Mark-Houwink plots The Mark-Houwink plot is used to compare the different structures of polymers It is often used to study branching Pectin HPC Dextran Pullulan Gum Arabic Drug Delivery Polymer Viscometer confirms drug attachment to polymer (lower IV) Parallel Mark-Houwink plot shows no molecular weight selectivity Relative Response Log[Intrinsic Viscosity] MH Plots of Polymer and Adduct Polymer Drug Adduct Log(Molecular Weight) Solvent: 0.1M Na 2 SO 4 Column: TSK PWXL Linear Concentration: 3 mg/ml Flowrate: 0.5 ml/min Inj. Vol.: ul dn/dc: Retention Volume (ml) 29

30 PLGA overlays 50:50, 65:35 and 75:25 Mark-Houwink plot IV versus Mw Structure by Mark Houwink plot - LHA and XHA 100 Intrinsic Viscosity (dl/g) Molecular Viscometer density LHA Molecular Light scattering weight XHA x x x10 Molecular Weight (Da) _23;49;47_HA_11004_ vdx : Intrinsic Viscosity (dl/g) _01;52;16_HA_11004_ vdx : Intrinsic Viscosity (dl/g) _21;57;07_XHA_1_1 17_07_2013_ vdx : Intrinsic Viscosity (dl/g) _22;58;21_XHA_1_1 17_07_2013_ vdx : Intrinsic Viscosity (dl/g) x10 30

31 Varying the Reaction Mixture Intrinsic Viscosity (dl/g) x x Molecular Weight (Da) _19;51;16_XHA_1_1 24_07_2013_ vdx : Intrinsic Viscosity (dl/g) _01;58;38_XHA_1_1 MIX_75_ vdx : Intrinsic Viscosity (dl/g) _15;14;34_XHA_1_1_idrat vdx : Intrinsic Viscosity (dl/g) _03;57;16_HA_lot_11004_ vdx : Intrinsic Viscosity (dl/g) 6 2x x10 Reaction Vessel Repeatability Structural Comparison Intrinsic Viscosity (dl/g) x x x10 Molecular Weight (Da) x _11;35;09_XHA_1_1 17_07_2013_ vdx : Intrinsic Viscosity (dl/g) _13;37;33_XHA_1_1 17_07_2013_ vdx : Intrinsic Viscosity (dl/g) _14;38;46_XHA_1_1 24_07_2013_ vdx : Intrinsic Viscosity (dl/g) _16;41;11_XHA_1_1 24_07_2013_ vdx : Intrinsic Viscosity (dl/g) _17;42;24_XHA_1_1 31_07_2013_ vdx : Intrinsic Viscosity (dl/g) _19;44;51_XHA_1_1 31_07_2013_ vdx : Intrinsic Viscosity (dl/g) 31

32 Retention Volume (ml) Retention Volume (ml) Chitosan In 95/5 Water/Acetic Acid with no salt at 30ºC: Native Chitosan N-(4-methylbenzyl) Chitosan Quat-188 Refractive Index Response (mv) Right Angle Light Scattering Response (mv) Viscometer DP Response (mv) N-(4-nitrobenzyl) Chitosan Quat-188 Refractive Index Response (mv) Right Angle Light Scattering Response (mv) Viscometer DP Response (mv) Native Chitosan N-(4-methylbenzyl) Chitosan Quat-188 N-(4-nitrobenzyl) Chitosan Quat- 188 Mn 48,706 11,571 20,514 Mw 219,718 28,606 64,733 Mz 2,520,333 97, ,845 [ή] Rh dn/dc Chitosan In 95/5 Water/Acetic Acid with no salt at 30ºC: _09;04;17_SAMPLE_1_01.vdt / Method: LSU-0003.vcm _09;04;17_SAMPLE_1_01.vdt : LSU-0003.vcm _10;05;16_SAMPLE_1_02.vdt : LSU-0003.vcm _11;06;15_SAMPLE_1_03.vdt : LSU-0003.vcm _12;07;14_SAMPLE_2_01.vdt : LSU-0004.vcm _13;08;13_SAMPLE_2_02.vdt : LSU-0004.vcm _23;03;18_SAMPLE_2_01.vdt : LSU-0004.vcm _23;54;24_SAMPLE_3_01.vdt : LSU-0005.vcm _00;45;24_SAMPLE_3_02.vdt : LSU-0005.vcm (1) _01;36;25_SAMPLE_3_03.vdt : LSU-0005.vcm a=0.829 (2) (1) Native Chitosan (2) N-(4-methylbenzyl) chitosan Quat-188 (3) N-(4-nitrobenzyl) chitosan Quat Log Molecular Weight (3) a =0.805 a= a=

33 CONCLUSIONS Traditional SEC with single concentration detector provides limited information The addition of a light scattering gives Absolute Molecular Weight / distributions/aggregation The viscometer gives Intrinsic Viscosity (density in solution) Triple Detection combines it all Absolute Molecular Weight / distributions Size (Rg/ Rh) / distributions Molecular Structure/Conformation (Mark Houwink) Conjugate Analysis A powerful tool in method development 33

34 Thank you for your attention! 34