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![Log P and Log D
• Log P, KOW – Partition Coefficient
ionisedun
wat
oct
watoct
solute
solute
P loglog /
• Log D Distribution coefficient
octsolute
D loglog• Log D – Distribution coefficient
pK log of eq ilibri m constant for acid dissociation
neutral
wat
ionised
wat
watoct
solutesolute
D loglog /
• pKa – log of equilibrium constant for acid dissociation
AaqOHOHHA )(
]][[ 3 AOH
K
• pKb – log of equilibrium constant for base dissociation
AaqOHOHHA )(32
][
3
HA
Ka
b
BHaqOHOHB )(2
][
]][[
B
BHOH
Kb
3
][B](https://image.slidesharecdn.com/stationaryandmobilephaseselectionmabphgradientanalysis33974-150324131049-conversion-gate01/85/Stationary-and-mobile_phase_selection_m_ab_ph_gradient_analysis_33974-3-320.jpg)
![Buffer Development Strategy: MES-MOPS-TAPS-CAPSO
Buffer Cocktail
• Replace cationic buffer
components with zwitterionic 10.510.5
components with zwitterionic
buffer species (Good’s Buffers)
• These buffer species contain one
y= 0.1577x + 4.9755
R²= 0.9996
8 5
9.5
value
y= 0.1577x + 4.9755
R²= 0.9996
8 5
9.5
value
p
quaternary amine group and one
sulfonic acid group. They do not bind
to the stationary phase in the
pH range of 6 10
7.5
8.5
MeasuredpH
Measured Value
Linear(MeasuredValue)7.5
8.5
MeasuredpH
Measured Value
Linear(MeasuredValue)
pH range of 6-10.
• They are not repelled by the stationary
phase so they can buffer the
5.5
6.5
0 10 20 30 40
Retention Time [min]
5.5
6.5
0 10 20 30 40
Retention Time [min]
stationary phase.
Retention Time [min]Retention Time [min]
MES MOPS TAPS CAPSO
42
6.1 7.2 8.4 9.6](https://image.slidesharecdn.com/stationaryandmobilephaseselectionmabphgradientanalysis33974-150324131049-conversion-gate01/85/Stationary-and-mobile_phase_selection_m_ab_ph_gradient_analysis_33974-42-320.jpg)
![Protein Standards Using Linear pH Gradient
60.0
7.55
93
40 0
50.0
-6.04
en-15.97-7
2.00-8.53
C-31.55-9.
30.0
40.0
bance[mAU]
ectin-1-5.87
97-6.20
8.18-6.37
Trypsinog
ucleaseA-22
CytochromeC
10.0
20.0
Absorb
Le
Lectin-2-6.9
Lectin-3-
Ribonu
C
0 5 10 15 20 25 30 35 40
-5.0
0 5 10 15 20 25 30 35 40
Retention Time [min]
44](https://image.slidesharecdn.com/stationaryandmobilephaseselectionmabphgradientanalysis33974-150324131049-conversion-gate01/85/Stationary-and-mobile_phase_selection_m_ab_ph_gradient_analysis_33974-44-320.jpg)
![mAb Charge Variant Separation, 0–100% B
100% B0% B
40.0 10.50
30.0
9.00
mAU]
pH trace(a)
20.0
7 00
8.00
bsorbance[m
10.0
6.00
7.00
Ab
0 5 10 15 20 25 30 35 40
-5.0 5.00
Retention Time [min]
*The pH trace at elution was obtained with the Thermo Scientific™ Dionex™ UltiMate™ 3000 pH and Conductivity
47
The pH trace at elution was obtained with the Thermo Scientific™ Dionex™ UltiMate™ 3000 pH and Conductivity
Monitoring Module (PCM-3000)](https://image.slidesharecdn.com/stationaryandmobilephaseselectionmabphgradientanalysis33974-150324131049-conversion-gate01/85/Stationary-and-mobile_phase_selection_m_ab_ph_gradient_analysis_33974-47-320.jpg)
![mAb Charge Variant Separation, 0–50% B
0% B 50% B
25.0 8.50
H t
20.0
mAU]
(b)
pH trace
10.0
7.00
bsorbance[m
0.0
6.00
Ab
0 5 10 15 20 25 30 35 40
-5.0 5.00
Retention Time [min]
48](https://image.slidesharecdn.com/stationaryandmobilephaseselectionmabphgradientanalysis33974-150324131049-conversion-gate01/85/Stationary-and-mobile_phase_selection_m_ab_ph_gradient_analysis_33974-48-320.jpg)
![mAb Charge Variant Separation, 25–50% B
25% B 50% B
16.0 8.00
10 0
7.75
mAU]
(c) pH trace
5.0
10.0
7.25
7.50
bsorbance[m
5.0
7.00
Ab
0 5 10 15 20 25 30 35 40
-2.0 6.60
Retention Time [min]
49](https://image.slidesharecdn.com/stationaryandmobilephaseselectionmabphgradientanalysis33974-150324131049-conversion-gate01/85/Stationary-and-mobile_phase_selection_m_ab_ph_gradient_analysis_33974-49-320.jpg)
![Repeat Injections of Ribonuclease A: Over 300 Runs
60 10.50
H t
Retention time reproducibility <0.8% RSD
25
9.00
pH trace
0
8.00
9.00
Run #300
nce[mAU]
50
-25
7.00
Run #200
Absorba
-75
-50
6.00
Run #100
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0
-100 5.00
Run #5
55
Retention Time [min]](https://image.slidesharecdn.com/stationaryandmobilephaseselectionmabphgradientanalysis33974-150324131049-conversion-gate01/85/Stationary-and-mobile_phase_selection_m_ab_ph_gradient_analysis_33974-55-320.jpg)
Uploaded byChromatography & Mass Spectrometry Solutions
Stationary and mobile_phase_selection_m_ab_ph_gradient_analysis_33974
The document discusses key considerations for selecting stationary and mobile phases in liquid chromatography, including the importance of chemical properties like log P, log D, and pKa. It outlines different chromatography modes such as reversed phase, normal phase, size exclusion, and hydrophilic interaction chromatography (HILIC), detailing their applications and advantages. Additionally, it covers various factors affecting separation efficiency like temperature, flow rate, and mobile phase composition.
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Similar to Stationary and mobile_phase_selection_m_ab_ph_gradient_analysis_33974
Stationary and mobile_phase_selection_m_ab_ph_gradient_analysis_33974
- 1. Stationary Phase andMobile Phase Selection for Liquid Chromatography Shanhua Lin PhD Research Scientist Tony Edge Scientific AdvisorScientific Advisor October 2014
- 2. Introductions Ch i lti f l l• Chemical properties of your molecule • Understanding the importance of log P, log D, pKa • Chromatography Mode SelectionChromatography Mode Selection • Reversed Phased • SEC N l Ph• Normal Phase • HILIC • Mixed Mode • Mobile phase considerations • Buffer selection Organic solvent selection• Organic solvent selection • Linear pH gradient for monoclonal antibodies charge variant analysis. • Platform method. • Fast analysis within 10 min. • mAb pI prediction. 2
- 3. Log P andLog D • Log P, KOW – Partition Coefficient ionisedun wat oct watoct solute solute P loglog / • Log D Distribution coefficient octsolute D loglog• Log D – Distribution coefficient pK log of eq ilibri m constant for acid dissociation neutral wat ionised wat watoct solutesolute D loglog / • pKa – log of equilibrium constant for acid dissociation AaqOHOHHA )( ]][[ 3 AOH K • pKb – log of equilibrium constant for base dissociation AaqOHOHHA )(32 ][ 3 HA Ka b BHaqOHOHB )(2 ][ ]][[ B BHOH Kb 3 ][B
- 4. Acid / BaseEquilibria NH2 NH3 +O OH O O - h i li 4 www.chemicalize.org
- 5. Dependence of RetentionFactor on pH 100 Mobile Phase: 35% MeCN, 65% 20 mM Buffer Hypersil GOLD 100 x 2.1mm 1010 Logk 1 L 0.1 0 2 4 6 8 10 12 14 pHp Acetaminophen Ibuprofen Nortriptyline Lidocaine Doxepin Imipramine p-Toluamide 5
- 6. The Impact ofSelectivity on Resolution Efficiency SelectivityRetentionEfficiency SelectivityRetention 2.5 3 Fixed values: N- 5000 k’ 5 1 .. ' ' 22 kN R 2 tion k’-5 α-1.05 N . 1 . 4 ' 2 k R ' k 1 1.5 Resolut k’' 1 2 k k 0.5 1 RSelectivity (α) has the greatest impact on 1.00 1.05 1.10 1.15 1.20 1.25 N 1.00 1.05 1.10 1.15 1.20 1.25 N 0 g p improving resolution 0 5000 10000 15000 20000 25000 0 5 10 15 20 25 N k 0 5000 10000 15000 20000 25000 0 5 10 15 20 25 N k Stationary phase, gradient delay volume, mobile phase, pressure / flow rate, 6 Stationary phase, gradient delay volume, mobile phase, pressure / flow rate, temperature affect selectivity
- 7. Column Selection –Basics Need retention between analyte and column • Mainly reverse phase, hydrophobic interactionsy p , y p • More polar compounds – weaker retention Column needs to differentiate between similar molecules ff• difficult to judge this as tend to be looking at very small differences Column needs to be stable in conditions being used • OverloadingOverloading • pH effects • Temperature effects 7 p
- 8. Reversed Phase Chromatography •Most popular form of chromatography • C18 phase 80-90% use • Non-Polar stationary phase or substrate, typically ODSy p yp y • Alkyl chain phases, phenyl, cyano, PFP, • Polar mobile phase; water / methanol / THF / ACN • Degree of retention is based primarily on hydrophobicity of moleculeg p y y p y 8
- 9. Reversed Phased Chromatography Bondedphase: • Endcapped Embedded C N O • Embedded • C18, C8, C4 etc. • Phenyl N y • TMS modified N O O Silica support: • Silica metal ion content O • Silica metal ion content • Totally porous, non-porous or superficially porous support • Pure silica or organic / inorganic hybrid • Particle size and particle size distribution • Pore size, surface area • Deactivation / nature of the end capping reagent 9 • Deactivation / nature of the end-capping reagent
- 10. Types of SilanolGroups – Secondary Interactions OHHO GeminalAnionic exchange site Si Surface Si O Siloxane exchange site Si OH M+ Free Surface metal Silica Si Si OH M+ Si Free Metal Silica particle Si Si Si HO activated OHOH HO Associated / Vicinal 10 Hydrogen bond
- 11. Stationary Phase Characterization •Hydrophobic retention (HR) Hydrophobic Interactions y p ( ) • k’ of neutral compound • Hydrophobic selectivity (HS) • α two neutral compounds that have different log P • Steric Selectivity (SS) • α sterically different moleculesα sterically different molecules • Hydrogen bonding capacity (HBC)y g g y ( ) • α molecule that hydrogen bonds and a reference • Good measure of degree of endcapping 11 • Gives indication of available surface area
- 12. Stationary Phase Characterization •Activity towards bases (BA) Interactions with Bases and Chelators • Activity towards bases (BA) • k’, tailing factor (tf) of strong base • Indicator of free silanols • Activity towards chelators (C) • k’, tailing factor (tf) of chelator • Indicator of silica metal content 12
- 13. Stationary Phase Characterization Interactionswith Acids and Ion Exchanges • Activity towards acids (AI) • k’, tf acid • Indicator of interactions with acidic compounds• Indicator of interactions with acidic compounds • Ion Exchange Capacity (IEX pH 7.6)g p y ( p ) • α base / reference compound • Indicator of total silanol activity • All silanols above pKa I E h C it (IEX H 2 7)• Ion Exchange Capacity (IEX pH 2.7) • α base / reference compound • Indicator of acidic silanol (SiO-) activity 13 • Indicator of acidic silanol (SiO ) activity
- 14. Column Characterization (Visualization) AC18 A PFP HR /10 HSAI Accucore C18 HR /10 HSAI Accucore PFP SSIEX (2.7) SSIEX (2.7) HBC IEX (7.6)BA C HBC IEX (7.6)BA C htt // / /USPNF/ l DB ht l 14 http://www.usp.org/app/USPNF/columnsDB.html
- 15. Using Selectivity toDesign a Separation 500 mAU 1,2,3 curcuminoids 2 00 2.50 HR /10 HSAI 0.50 1.00 1.50 2.00 HS SSIEX (2.7) AI Accucore C18 Solid Core C18 Accucore Polar Premium 1 0.00 HBCC Accucore Polar Premium Accucore Phenyl-Hexyl 2 3 Polar Premium shows different selectivity and separates the peaks IEX (7.6)BA 0.0 1.0 2.0 3.0 0 Minutes 15 Removing uncertainty by applying science to Sample preparation
- 16. Size Exclusion Chromatography Sll l l tSmall molecules can enter pores, Large molecules cannot 16
- 17. SEC Columns • Moleculesare eluted based on their size in solution • Linear or rod-like molecules will elute before globular molecules of the same MW • Resolution is determined by the volume of pores with diametersResolution is determined by the volume of pores with diameters between the inclusion and exclusion limits of the solutes • Mobile phases should be selected to minimize interaction with the chromatographic surface Molecular Weight (kDaltons)g ( ) Pore Size Proteins Pullulans PEOs/PEGs 60Å 0.1-6 0.3-6 0.1-4 Å120Å 0.1-50 0.3-12 0.4-10 300Å 1-500 1-100 2-100 1000Å 20-4000 20->1000 Not recommended 17
- 18. Typical Compounds Separatedusing SEC SEC / GPC separates analytes based on their size • Protein mixtures • Used for purification • Used for identification • Sample pretreatment • Orthoganol separation, used in bioanlaysis P t h i l• Petrochemical • Identification of polymers 18
- 19. Polyethylene Oxides/Glycols Columns: BioBasicSEC, 5µm, 300x7.8mm Eluent: 100% water Flow: 1 0 mL/min MW 1. 965,000 2. 4,120 3 1 900 Comparison of Pore Size Flow: 1.0 mL/min Detector: ELSD 3. 1,900 4. 1,080 5. 106 3 300Å 100060Å 2 1 5 120Å 1 3 4 300Å 2 3 4 5 1000 Å 1 + 5 2 4 5 1 Time - Minutes 0 1 2 3 4 5 6 7 8 9 10111213 Time - Minutes 0 1 2 3 4 5 6 7 8 9 10111213 0 1 2 3 4 5 6 7 8 9 10 11 12 Time - Minutes Time - Minutes 0 2 4 6 8 10 12 14 16 19 Time - Minutes Time Minutes
- 20. Advantages and Disadvantagesof SEC • Advantages • It separates based on size• It separates based on size • Possible to separate different shaped molecules • Very useful for preparative scale chromatographyy p p g p y • Ideal for coarse separations of analytes • Disadvantages • The resolution tends to be very poor N d t th t th i t ti ith th t ti• Need to ensure that there are no interactions with the stationary phase and the analytes • Does not allow a full separation over a very large scalep y g • Materials designed to work over a limited analyte size • Not applicable to small molecules 20
- 21. Normal Phase Chromatography •Analyte displaces solvent molecules from the silica surface Solvent molecules molecules from the silica surface • Eluting properties of solvent are Analyte g p p based on hydrogen bonding interactions • Water is a strong solvent, hexane is weak Polar Stationary Phase Non-Polar Mobile Phase 21
- 22. Typical Compounds Separatedusing NPC • Sugar Analysis • Molecules very polar and ideally suited to NPCMolecules very polar and ideally suited to NPC • Useful in the field of biological sciences • Protein and Peptide Analysis • Identification and quantificationIdentification and quantification • Steroid analysis• Steroid analysis • Identification and quantification • Fat soluble vitamins • Compounds not soluble in aqueous mobile phases 22 • Compounds not soluble in aqueous mobile phases
- 23. Hypersil GOLD Silica– Steroids (NP) 70 1 Column: Hypersil GOLD Silica Analytes: 1. Progesterone 60 5 µm, 150 mm x 4.6 mm (i.d)2. 21-Hydroxyprogesterone-21-acetate 3. 17-α-Hydroxyprogesterone 4. Cortisone s 40 50 2 Chromatographic conditions: Mobile phase - 19:1 (v/v) n-C6H14/EtOH Flow rate - 1.5 ml min-1 5. 11-α-Hydroxyprogesterone 6. Corticosterone 7. Hydrocortisone mVolt 30 3 5 Temperature - 30 °C Detection - UV (254 nm) Injection volume - 5 µl 10 20 4 6 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 23 Minutes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
- 24. Advantages and Disadvantagesof Normal Phase Chromatography • Advantages Id l f l d• Ideal for very polar compounds • Ideal for water insoluble compounds • Disadvantages • Not to be used with non-polar compounds • Mobile phases tend to be very flammable • E.g. Hexane • Little selectivity options 24
- 25. HILIC • Hydrophilic stationaryphase with aqueous (5- 40%) / organic (95 60%) mobile phase40%) / organic (95-60%) mobile phase • Enhanced sensitivity in MS • Water forms a polar layer semi-immobilized onto• Water forms a polar layer semi-immobilized onto the surface of the stationary phase • Polar analytes partition into aqueous layer andPolar analytes partition into aqueous layer and are retained longer • partitioning R R R R I I I I O O O O p g • hydrogen bonding • weak electrostatic interactions O O O O I I I I Si Si Si Si • Differences in stationary phase will affect retention 25
- 26. HILIC retention behaviourof polar analytes Effect of % organic on capacity factor 2.00 2.50 1.00 1.50 k Uracil Cytosine 0.00 0.50 50 60 70 80 90 10050 60 70 80 90 100 % MeCN Column: Hypersil GOLD HILIC150 x 4.6 mm, 5 µm Mobile phase: 10mM Ammonium Acetate, pH 5.0 / MeCN Flow rate: 0.6 mL/min Detection: UV at 254 nm Temperature: 30 °C 26
- 27. HILIC: Improved MSsensitivity with MS detection SN: 35 100 SN: 551 m/z = 162 6 163 6 Reversed-phase HILIC 40 60 80 100 tiveAbundance m/z = 162.6 - 163.6 Nicotine 40 60 80 100 RelativeAbundance m/z = 162.6 - 163.6 Nicotine 15x sensitivity 80 100 20 40 Relat SN: 15 m/z = 176.7 - 177.7 Cotinine 80 100 20 R SN: 80 m/z = 176.7 - 177.7 Cotinine 5x sensitivity 0 20 40 60 20 40 60 5x sensitivity 1.0 2.0 3.0 4.0 Time (min) 0 0.0 1.0 2.0 3.0 4.0 5.0 Time (min) Column: Hypersil GOLD 150 x 2.1 mm 5µm Column: Hypersil GOLD HILIC, 150 x 2.1 mm 5µm M bil h A i f t 50 M H 3 5/ M CNMobile phase: H2O/ MeCN (98:2) + 0.1% formic acid Detection: +ESI (spray conditions adjusted for higher aqueous content of mobile phase) Injection: 1 ng on column Mobile phase: Ammonium formate 50 mM pH 3.5/ MeCN (10:90) Detection: +ESI Injection: 1 ng on column 27
- 28. Classification of HILICphases • Radar plots allow visual assessment and quick comparison of HILIC stationary phases k U k Uridine α (CH2) idi / 5 th l idi α (CH2) Syncronis HILIC (5 µm) α (CH2) α uridine / 5‐methyluridine α (OH) α uridine / 2’‐deoxyuridine ( / ) id bi / d i 0.5 1.0 α (CH2) α (OH)k uridine α (V/A) α vidarabine / adenosine α (2dG/3dG) α 2’‐deoxyguanosine / 3’‐deoxyguanosine 0.0 α (V/A)α (Tb/Tp) α (AX) α SPTS / Uracil α (CX) α TMPAC / Uracil α (2dG/3dG)α (CX) α (Tb/Tp) α theobromine / theophylline α (AX) 28
- 29. HILIC tests -Results 1.0 α (CH2) (OH)k idi Syncronis HILIC 1.0 α (CH2) Acclaim HILIC-10 1.0 α (CH2) Hypersil GOLD HILIC 0.0 0.5 α (OH) α (V/A)α (Tb/Tp) k uridine 0.0 0.5 α (OH) α (V/A)α (Tb/Tp) k uridine 0.0 0.5 α (OH) α (V/A)α (Tb/Tp) k uridine α (2dG/3dG) α (AX) α (CX) α (2dG/3dG) α (AX) α (CX) α (2dG/3dG) α (AX) α (CX) α (CH2) Accucore HILIC 1 0 α (CH2) Syncronis Silica α (CH2) Hypersil GOLD Silica 0.0 0.5 1.0 α (CH2) α (OH) α (V/A)α (Tb/Tp) k uridine 0.0 0.5 1.0 α (OH) α (V/A)α (Tb/Tp) k uridine 0.0 0.5 1.0 α (C ) α (OH) α (V/A)α (Tb/Tp) k uridine ( ) α (2dG/3dG) α (AX) α (CX) ( p) ( ) α (2dG/3dG) α (AX) α (CX) ( )0.0 α (V/A) α (2dG/3dG) α (AX) α (CX) α (Tb/Tp) 29 ( ) ( )α ( )
- 30. Mixed-Mode Chromatography • Definition •Hydrophobic interaction + ion-exchange interactionHydrophobic interaction + ion exchange interaction • Benefits • Adjustable selectivity • Simplified mobile phase (no ion-pairing reagents) • Simultaneous separation of different types of analytes T• Types • Anion-exchange/reversed-phase (AEX/RP) • Cation-exchange/reversed-phase (CEX/RP)g p ( ) • Anion-exchange/cation-exchange/reversed-phase (AEX/CEX/RP) Me O N N MeH N OH O Acclaim Mixed-Mode WAX-1 Acclaim Mixed-Mode WCX-1 30 O H Acclaim Mixed Mode WCX 1
- 31. Selectivity Adjusted byIonic Strength Column: AcclaimMixed-Mode WAX-1, 5 µm100 mM N N Me O MeH µ Dimension: 4.6 x 150 mm Mobile Phase: 50/50 v/v acetonitrile/phosphate buffer Temperature: 30 °C 1 2 Phosphate buffer, pH 6 Flow Rate: 1 mL/min Inj. Volume: 2 µL Detection: UV (210 nm)AU 1 Peaks: 1. Butylbenzene (0.1 mg/mL) 2. 4-Hydroxybenzoic acid (0.5 mg/mL) 1 2 20 mM Phosphate buffer, pH 6 CO2H Butylbenzene 4-Hydroxybenzoic acid 0 7.5 15 Minutes OH 31
- 32. Selectivity Adjusted bypH Column: Acclaim Mixed-Mode WAX-1, 5 µm 1 N N Me O MeH µ Dimension: 4.6 x 150 mm Mobile Phase: 50/50 v/v acetonitrile/ 20 mM phosphate buffer Temperature: 30 °C2 1 pH 6.0 Flow Rate: 1 mL/min Inj. Volume: 2 µL Detection: UV (210 nm) 2 Peaks: 1. Butylbenzene (0.1 mg/mL) 2. 4-Hydroxybenzoic acid (0.5 mg/mL) 2 1 pH 2.6 AU CO2H 0 7.5 15 Minutes Butylbenzene 4-Hydroxybenzoic acid OH 32
- 33. Selectivity Adjusted byOrganic Content 50% Acetonitrile N N Me O MeH50% Acetonitrile 1 2 Column: Acclaim Mixed-Mode WAX-1, 5 µm Dimension: 4 6 x 150 mm H 2 Dimension: 4.6 x 150 mm Mobile Phase: Acetonitrile/ 20 mM phosphate buffer, pH6 Temperature: 30 °C Flow Rate: 1 mL/minAU 1 2 o ate / Inj. Volume: 2 µL Detection: UV (210 nm) Peaks: 1. Butylbenzene (0.1 mg/mL) AU 45% Acetonitrile 2 y ( g ) 2. 4-Hydroxybenzoic acid (0.5 mg/mL) CO2H 0 10 20 Minutes Butylbenzene 4-Hydroxybenzoic acid OH 33
- 34. Effect of IonicStrength on the Efficiency (N) • For ionic analytes, higher ionic strength mobile phases increased efficiency as there is a lower ion exchange interaction • This is due to the competitive nature of the buffer for the ionic sites on the silica surface. • Increased ionic strength leads to a reduced ion exchange separation mechanism t ib ti thi l ti ti d b diff t f diff t lcontribution, this elution time decreases may be different for different sample components. • Changing the buffer concentration may result in resolved peaks to co-elute C l ti k b l d t diff t b ff t ti• Co-eluting peaks may be resolved at a different buffer concentration • If the buffer concentration is too low, it will not be able to act as a buffer. B ff h ld b t t 5 M l• Buffer should be present at > 5 mMol. • If the buffer concentration is too high • the eluent solution becomes viscous • Ion suppression with MS detection • UV absorbance with some buffers 34 • solubility of the buffer with organic solvent becomes problematic
- 35. Mobile Phase –Addition of Buffer Buffer pKa pH Range Phosphate 2.1 1.1 – 3.1 7.2 6.2 – 8.2 12.3 11.3 – 13.3 Citrate 3.1 2.1 – 4.1 4.7 3.7 – 5.7 5 4 4 4 6 45.4 4.4 – 6.4 Formate 3.8 2.8 – 4.8 Acetate 4.8 3.8 – 5.8 Tris (hydroxymethyl) aminomethane 8.3 7.3 – 9.3 A i 9 2 8 2 10 2Ammonia 9.2 8.2 – 10.2 Borate 9.2 8.2 – 10.2 Diethylamine 10.5 9.5 – 11.5 35 Diethylamine 10.5 9.5 11.5
- 36. Use of IonPairing Reagents 1) Procainamide, 2) N-Acetyl procainamide, 3) N-propionyl procainamide Absorvance(mAU) 0.05% TFA1 0.3% TFA1 3 Absorvance(mAU) Mobile phase: 2 3 2 Aqueous is water containing 0.05, 0.3, 0.5 or 1%TFA Organic is acetonitrile/2 -propanol (1 3) t i i 0 05 0 3 0 5 Time (min) 0 2.5 5 7.5 Time (min) 0 2.5 5 7.5 mAU) 3 1 0.5% TFA AU) 1.0% TFA3 (1:3) containing 0.05, 0.3, 0.5 or 1%TFA Gradient: 35 to 95% organic in 10 i Absorvance(m 2 Absorvance(mA 21 min Flow rate: 1 ml/min Detection: 270 nm Temperature: 50 °C 36 Time (min) 0 2.5 5 7.5 Time (min) 0 2.5 5 7.5
- 37. Mobile Phase Selectivity- Snyder Triangle Proton acceptor II M OHII MeOH III THF VI MeCN III III VI MeCN V IV III VIII VI VII DipoleProton Interactiondonor Solvents are chosen near the apexes of the triangle to obtain 37 the widest selectivity differences
- 38. Effect of OrganicSolvent Content on Solute Retention in RP Chromatography Solute 1 A linear relationship is observed when solute interaction with the stationary phase is predominantly via hydrophobic interactions Solute 2 Solute 3 log k 20% 40% 60% 80% 100% % Methanol 38
- 39. Regulatory Expectations forthe characterization of CQAs in monoclonal antibodies (mAbs) Protein Analytical Chemistry Techniques Used in the Testing of Biological Products Protein Property Characterization Batch Release/Stability Further Development of Assay Size Mass spec (intact mass) SDS-PAGE, SEC Impurity (aggregates, fragments) Charge CE-IEF, IEC, pH-IEC CE-IEF, IEC, pH-IEC Acylation, Deamidation, Sialylation variants Hydrophobicity peptide mapping, hydrophobic interaction chromatography (HIC) Deamidation, oxidation, (U)HPLC Concentration Amino acid analysis, HPLC method, ELISA UV A280 Carbohydrate analysis LC/MS, fluorescent labeling, monosaccharide composition HPAE-PAD (IC) (U)HPLC Heterogeneity 2°, 3° Structure Circular dichroism, peptide mapping Disulphide mapping Peptide Mapping LC/MS, N- C- sequencing AAA analysis (U)HPLC-FLD or (U)HPLC-CAD Binding activity ELISA, Biacore ELISA, Biacore Potency Cell-based assays Cell-based potency assay Identity Western blotting, peptide mapping, (U)HPLC Western blotting, peptide mapping, 39 Adapted from Camille Dycke et. al., GEN October 15, 2010
- 40. Protein and mAbSeparation on IEX Columns Salt Gradient pH Gradient • Most widely used method • Relatively simple to make • Can predict elution profile with pI value Relatively simple to make the buffer • Takes longer to optimize the • Lower salt concentration in collected fractions • Takes longer to optimize the separation condition (pH, salt concentration) • In many cases, improved resolution was observed • Difficult to generate a linear pH gradient 40
- 41. pH Gradient Buffers– How Do They Work? + Isoelectric Point (pI) Protein Elution Mechanisms on IEX + Buffer pH typically < pI Cation Exchange NH3 R + COO- Cationic protein binds to negatively charged cation exchanger + ++ ++ Buffer/System pH Cation-Exchange Chromatography NH3 R + COOH cation exchanger + ++ 0 5 6 7 8 9 10 11 12 u e /Sys e p Buffer pH typically > pI 4 Anionic protein - -- pH range covered by CX-1 pH gradient buffersBuffer pH typically pI Anion-Exchange Chromatography R COO- Anionic protein binds to positively charged anion exchanger - - - - - p g y p g – Protein net charge vs. pH NH2 R 41
- 42. Buffer Development Strategy:MES-MOPS-TAPS-CAPSO Buffer Cocktail • Replace cationic buffer components with zwitterionic 10.510.5 components with zwitterionic buffer species (Good’s Buffers) • These buffer species contain one y= 0.1577x + 4.9755 R²= 0.9996 8 5 9.5 value y= 0.1577x + 4.9755 R²= 0.9996 8 5 9.5 value p quaternary amine group and one sulfonic acid group. They do not bind to the stationary phase in the pH range of 6 10 7.5 8.5 MeasuredpH Measured Value Linear(MeasuredValue)7.5 8.5 MeasuredpH Measured Value Linear(MeasuredValue) pH range of 6-10. • They are not repelled by the stationary phase so they can buffer the 5.5 6.5 0 10 20 30 40 Retention Time [min] 5.5 6.5 0 10 20 30 40 Retention Time [min] stationary phase. Retention Time [min]Retention Time [min] MES MOPS TAPS CAPSO 42 6.1 7.2 8.4 9.6
- 43. Benefit of LinearpH Gradient: Generic Approach • A generic approach to charge variant analysis, covering the pH range 5 6 to 10 25.6 to 10.2 • The same pH gradients is applicable to majority of mAb charge variants with pI value between 6-10. • pI value of the unknown mAb can be predicted from the correlation curvepI value of the unknown mAb can be predicted from the correlation curve 43
- 44. Protein Standards UsingLinear pH Gradient 60.0 7.55 93 40 0 50.0 -6.04 en-15.97-7 2.00-8.53 C-31.55-9. 30.0 40.0 bance[mAU] ectin-1-5.87 97-6.20 8.18-6.37 Trypsinog ucleaseA-22 CytochromeC 10.0 20.0 Absorb Le Lectin-2-6.9 Lectin-3- Ribonu C 0 5 10 15 20 25 30 35 40 -5.0 0 5 10 15 20 25 30 35 40 Retention Time [min] 44
- 45. Linear Correlation ofElution pH vs pI 10.510.5 10 Protein standards mAb standards Cytochrome C y= 1.6923x - 7.2914 R²= 0.9929 9 9.5 10 Cytochrome C y= 1.6923x - 7.2914 R²= 0.9929 9 9.5 10 y= 1.1083x - 1.637 R²= 0.9988 9 9.5 e Ribonuclease A 8 8.5 9 redpHvalue Measured pH value Ribonuclease A 8 8.5 9 redpHvalue Measured pH value 8 8.5 utionpHvalue MAbElution pH value L ti 3 Trypsinogen 6.5 7 7.5 Measur Linear(MeasuredpH value) L ti 3 Trypsinogen 6.5 7 7.5 Measur Linear(MeasuredpH value) 7 7.5 MAbElu Linear(MAb Elution pH value) Lectin-1 Lectin-2 Lectin-3 5.5 6 7.5 8.5 9.5 10.5 Lectin-1 Lectin-2 Lectin-3 5.5 6 7.5 8.5 9.5 10.5 6 6.5 6.5 7.5 8.5 9.5 10.5 pI valuepI value MAb pI value 45
- 46. Benefit of LinearpH Gradient: Simple Optimization • The method can be simply optimized • By running a shallower pH gradient a higher resolution separation is obtained (e.g. 50-100%, rather than 0-100%B) 46
- 47. mAb Charge VariantSeparation, 0–100% B 100% B0% B 40.0 10.50 30.0 9.00 mAU] pH trace(a) 20.0 7 00 8.00 bsorbance[m 10.0 6.00 7.00 Ab 0 5 10 15 20 25 30 35 40 -5.0 5.00 Retention Time [min] *The pH trace at elution was obtained with the Thermo Scientific™ Dionex™ UltiMate™ 3000 pH and Conductivity 47 The pH trace at elution was obtained with the Thermo Scientific™ Dionex™ UltiMate™ 3000 pH and Conductivity Monitoring Module (PCM-3000)
- 48. mAb Charge VariantSeparation, 0–50% B 0% B 50% B 25.0 8.50 H t 20.0 mAU] (b) pH trace 10.0 7.00 bsorbance[m 0.0 6.00 Ab 0 5 10 15 20 25 30 35 40 -5.0 5.00 Retention Time [min] 48
- 49. mAb Charge VariantSeparation, 25–50% B 25% B 50% B 16.0 8.00 10 0 7.75 mAU] (c) pH trace 5.0 10.0 7.25 7.50 bsorbance[m 5.0 7.00 Ab 0 5 10 15 20 25 30 35 40 -2.0 6.60 Retention Time [min] 49
- 50. Benefit of LinearpH Gradient: Fast Analysis • By using • A smaller particle (5 µm rather than 10 µm)• A smaller particle (5 µm rather than 10 µm) • A shorter cation-exchange column (4 × 50 mm) • A high flow rate at 2 mL/min mAb charge variant profile can be quickly determined within 10 min. 50
- 51. mAb Charge VariantSeparation With Fast pH Gradient 140 11.00 (b) 0% B 100% B 100 120 10.00 pH trace (b) 80 9.00 ce(mAU) 40 60 7.00 8.00 Absorban 0 20 6.00 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 -20 5.00 Retention Time (min) 51
- 52. Benefit of LinearpH Gradient: High Resolution • In most cases, we observed improved separation of the charge variants over salt gradientover salt gradient. 52
- 53. Salt vs pHGradient IEC of mAb Sample 10.0 15.0 30.0 5.0 10.0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 0.0 min %B: 10.0 Salt gradient 10 0 15.0 50.0 5.0 10.0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 0.0 min %B: 25.0 25.0 pH gradient 53 30 min gradient, Thermo Scientific™ MabPac™ SCX-10, 10 µm, 4 × 250 mm column
- 54. Benefit of LinearpH Gradient: Great Precision • The retention times in pH gradient IEC are highly reproducible • This makes prediction of pI very consistent• This makes prediction of pI very consistent 54
- 55. Repeat Injections ofRibonuclease A: Over 300 Runs 60 10.50 H t Retention time reproducibility <0.8% RSD 25 9.00 pH trace 0 8.00 9.00 Run #300 nce[mAU] 50 -25 7.00 Run #200 Absorba -75 -50 6.00 Run #100 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 -100 5.00 Run #5 55 Retention Time [min]
- 56. Conclusions Ch i lti f l l• Chemical properties of your molecule • Understanding the importance of log P, log D, pKa • Chromatography Mode SelectionChromatography Mode Selection • Reversed Phased • SEC N l Ph• Normal Phase • HILIC • Mixed Mode • Mobile phase considerations • Buffer selection Organic solvent selection• Organic solvent selection • Linear pH gradient for monoclonal antibodies charge variant analysis. • Platform method. • Fast analysis within 10 min. • mAb pI prediction. 56
- 57.