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Stationary Phase and Mobile Phase Selection for Liquid Chromatography
Shanhua Lin PhDResearch Scientist
Tony EdgeScientific AdvisorScientific Advisor
October 2014
IntroductionsCh i l ti 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
Log P and Log D
• Log P, KOW – Partition Coefficient
ionisedun
wat
octwatoct solute
soluteP loglog /
• Log D Distribution coefficient
octsoluteD loglog• Log D – Distribution coefficient
pK log of eq ilibri m constant for acid dissociation
neutralwat
ionisedwat
watoct solutesoluteD loglog /
• pKa – log of equilibrium constant for acid dissociation
AaqOHOHHA )( ]][[ 3 AOHK
• pKb – log of equilibrium constant for base dissociation
AaqOHOHHA )(32 ][3
HAKa
b
BHaqOHOHB )(2 ][]][[
BBHOHKb
3
][B
Acid / Base Equilibria
NH2 NH3+O OH O O
-
h i li4
www.chemicalize.org
Dependence of Retention Factor on pH
100
Mobile Phase: 35% MeCN, 65% 20 mM BufferHypersil GOLD 100 x 2.1mm
1010
Log
k
1
L
0.10 2 4 6 8 10 12 14
pHpAcetaminophen Ibuprofen Nortriptyline LidocaineDoxepin Imipramine p-Toluamide
5
The Impact of Selectivity on Resolution
Efficiency SelectivityRetentionEfficiency SelectivityRetention 2.5
3
Fixed values:N- 5000k’ 5
1.. '
'22
kN
R2
tion
k’-5α-1.05
N.
1.
4 '2 k
R
'k 1
1.5
Res
olut
k’'1
2
kk
0.5
1RSelectivity (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 1.25N
0
g pimproving resolution 0 5000 10000 15000 20000 25000
0 5 10 15 20 25
Nk
0 5000 10000 15000 20000 25000
0 5 10 15 20 25
Nk
Stationary phase, gradient delay volume, mobile phase, pressure / flow rate,
6
Stationary phase, gradient delay volume, mobile phase, pressure / flow rate, temperature affect selectivity
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
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
Reversed Phased Chromatography
Bonded phase: • Endcapped
EmbeddedC
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
Types of Silanol Groups – Secondary Interactions
OHHO GeminalAnionicexchange site
SiSurface
Si O Siloxaneexchange site
Si OH
M+
Free
Surfacemetal
SilicaSi
Si OHM+
Si
Free
Metal
Silicaparticle
Si SiSi
HOactivated
OHOH
HOAssociated / Vicinal
10
Hydrogen bond
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
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
Stationary Phase Characterization
Interactions with 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
Column Characterization (Visualization)A C18 A PFP
HR /10
HSAI
Accucore C18HR /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 l14
http://www.usp.org/app/USPNF/columnsDB.html
Using Selectivity to Design a Separation
500mAU 1,2,3 curcuminoids
2 00
2.50HR /10
HSAI
0.50
1.00
1.50
2.00 HS
SSIEX (2.7)
AI
Accucore C18
Solid Core C18
Accucore Polar Premium1
0.00
HBCC
Accucore Polar Premium
Accucore Phenyl-Hexyl
23
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
Size Exclusion Chromatography
S ll l l tSmall molecules can enter pores, Large molecules cannot
16
SEC Columns• Molecules are 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
Typical Compounds Separated using 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
Polyethylene Oxides/Glycols
Columns: BioBasic SEC, 5µm, 300x7.8mmEluent: 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/minDetector: ELSD
3. 1,900 4. 1,080 5. 106
3 300Å 100060Å
21 5
120Å 13
4
300Å2 3 4 5 1000
Å 1 + 5
24
51
Time - Minutes0 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 12Time - Minutes Time - Minutes
0 2 4 6 8 10 12 14 16
19
Time - Minutes Time Minutes
Advantages and Disadvantages of 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
Normal Phase Chromatography
• Analyte displaces solvent molecules from the silica surface
Solvent moleculesmolecules from the silica surface
• Eluting properties of solvent are Analyte
g p pbased on hydrogen bonding interactions
• Water is a strong solvent, hexane is weak
Polar Stationary Phase Non-Polar Mobile Phase
21
Typical Compounds Separated using 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
Hypersil GOLD Silica – Steroids (NP)
701
Column:
Hypersil GOLD SilicaAnalytes:
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
mVo
lt
303
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
Minutes1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Advantages and Disadvantages of Normal Phase Chromatography
• AdvantagesId 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
HILIC
• Hydrophilic stationary phase 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
• partitioningR R R RI I I IO O O O
p g
• hydrogen bonding
• weak electrostatic interactions
O O O OI I I ISi Si Si Si
• Differences in stationary phase will affect
retention
25
HILIC retention behaviour of 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 / MeCNFlow rate: 0.6 mL/min Detection: UV at 254 nmTemperature: 30 °C
26
HILIC: Improved MS sensitivity with MS detection
SN: 35100
SN: 551m/z = 162 6 163 6
Reversed-phase HILIC
40
60
80
100
tive
Abu
ndan
ce
m/z = 162.6 - 163.6 Nicotine
40
60
80
100
Rel
ativ
e A
bund
ance
m/z = 162.6 - 163.6
Nicotine 15x sensitivity
80
100
20
40
Rel
at
SN: 15 m/z = 176.7 - 177.7 Cotinine80
100
20
R
SN: 80 m/z = 176.7 - 177.7 Cotinine
5x sensitivity
0
20
40
60
20
40
605x sensitivity
1.0 2.0 3.0 4.0
Time (min)
00.0 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 5µmM 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: +ESIInjection: 1 ng on column
27
Classification of HILIC phases
• 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 i0.5
1.0α (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 / Uracilα (2dG/3dG)α (CX)
α (Tb/Tp) α theobromine / theophyllineα (AX)
28
HILIC tests - Results
1.0α (CH2)
(OH)k idi
Syncronis HILIC
1.0α (CH2)
Acclaim HILIC-10
1.0α (CH2)
Hypersil GOLD HILIC
0.0
0.5α (OH)
α (V/A)α (Tb/Tp)
k uridine
0.0
0.5α (OH)
α (V/A)α (Tb/Tp)
k uridine
0.0
0.5α (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 0α (CH2)
Syncronis Silica
α (CH2)
Hypersil GOLD Silica
0.0
0.5
1.0α (CH2)
α (OH)
α (V/A)α (Tb/Tp)
k uridine
0.0
0.5
1.0α (OH)
α (V/A)α (Tb/Tp)
k uridine
0.0
0.5
1.0α (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 ( )
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)
MeO
N NMeH
NOH
O
Acclaim Mixed-Mode WAX-1
Acclaim Mixed-Mode WCX-1
30
OH
Acclaim Mixed Mode WCX 1
Selectivity Adjusted by Ionic Strength
Column: Acclaim Mixed-Mode WAX-1, 5 µm100 mM
N NMe
O
MeH
µDimension: 4.6 x 150 mmMobile Phase: 50/50 v/v acetonitrile/phosphate bufferTemperature: 30 °C
1
2
Phosphate buffer, pH 6
Flow Rate: 1 mL/minInj. Volume: 2 µLDetection: 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 acid0 7.5 15
Minutes
OH
31
Selectivity Adjusted by pH
Column: Acclaim Mixed-Mode WAX-1, 5 µm1
N NMe
O
MeH
µDimension: 4.6 x 150 mmMobile Phase: 50/50 v/v acetonitrile/ 20 mM phosphate bufferTemperature: 30 °C2
1pH 6.0
Flow Rate: 1 mL/minInj. Volume: 2 µLDetection: UV (210 nm)2Peaks: 1. Butylbenzene (0.1 mg/mL)
2. 4-Hydroxybenzoic acid (0.5 mg/mL)
2
1 pH 2.6AU
CO2H
0 7.5 15Minutes
Butylbenzene 4-Hydroxybenzoic acidOH
32
Selectivity Adjusted by Organic Content
50% AcetonitrileN N
MeO
MeH50% Acetonitrile1
2
Column: Acclaim Mixed-Mode WAX-1, 5 µmDimension: 4 6 x 150 mm
H
2 Dimension: 4.6 x 150 mmMobile Phase: Acetonitrile/ 20 mM phosphate buffer, pH6Temperature: 30 °CFlow Rate: 1 mL/minAU
1
2
o ate /Inj. Volume: 2 µLDetection: UV (210 nm)Peaks: 1. Butylbenzene (0.1 mg/mL)
AU
45% Acetonitrile
2y ( g )
2. 4-Hydroxybenzoic acid (0.5 mg/mL)
CO2H
0 10 20Minutes Butylbenzene 4-Hydroxybenzoic acid
OH
33
Effect of Ionic Strength 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
Mobile Phase – Addition of Buffer
Buffer pKa pH RangePhosphate 2.1 1.1 – 3.1
7.2 6.2 – 8.212.3 11.3 – 13.3
Citrate 3.1 2.1 – 4.14.7 3.7 – 5.75 4 4 4 6 45.4 4.4 – 6.4
Formate 3.8 2.8 – 4.8Acetate 4.8 3.8 – 5.8Tris (hydroxymethyl) aminomethane
8.3 7.3 – 9.3
A i 9 2 8 2 10 2Ammonia 9.2 8.2 – 10.2Borate 9.2 8.2 – 10.2Diethylamine 10.5 9.5 – 11.5
35
Diethylamine 10.5 9.5 11.5
Use of Ion Pairing Reagents
1) Procainamide, 2) N-Acetyl procainamide, 3) N-propionyl procainamide
Abs
orva
nce
(mA
U)
0.05% TFA1 0.3% TFA1
3
Abs
orva
nce
(mA
U)
Mobile phase:
2
3
2Aqueous 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
mA
U)
31
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
Abs
orva
nce
(m
2 Abs
orva
nce
(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
Mobile Phase Selectivity - Snyder Triangle
Proton acceptor
II M OHII MeOHIII THFVI MeCN
III
IIIVI MeCN
V
IVIII
VIII VI
VIIDipoleProtonInteractiondonor
Solvents are chosen near the apexes of the triangle to obtain
37
the widest selectivity differences
Effect of Organic Solvent Content on Solute Retention in RP Chromatography
Solute 1A linear relationship is observed whensolute interaction with the stationary phase is predominantly via hydrophobic interactions
Solute 2
Solute 3
log k
20% 40% 60% 80% 100%% Methanol
38
Regulatory Expectations for the 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, monosaccharidecomposition
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
Protein and mAb Separation 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
pH Gradient Buffers – How Do They Work?
+IsoelectricPoint (pI)
Protein Elution Mechanisms on IEX
+Buffer pH typically < pI
Cation Exchange
NH3R +
COO -
Cationic protein binds to
negatively charged cation exchanger
+ ++
++
Buffer/System pH
Cation-ExchangeChromatography
NH3R +
COOH
cation exchanger
+ ++
05 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-ExchangeChromatography
R
COO -
Anionic proteinbinds to
positively chargedanion exchanger
- -
- - -
p g y p g
–Protein net charge vs. pH
NH2R
41
Buffer Development Strategy: MES-MOPS-TAPS-CAPSO Buffer Cocktail
• Replace cationic buffer components with zwitterionic 10.510.5components with zwitterionicbuffer species (Good’s Buffers)
• These buffer species contain one
y = 0.1577x + 4.9755R² = 0.9996
8 5
9.5
va
lue
y = 0.1577x + 4.9755R² = 0.9996
8 5
9.5
va
lue
pquaternary 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
Me
as
ure
d p
H
Measured ValueLinear (Measured Value)7.5
8.5
Me
as
ure
d p
H
Measured ValueLinear (Measured Value)
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 40Retention Time [min]
5.5
6.5
0 10 20 30 40Retention Time [min]
stationary phase.Retention Time [min]Retention Time [min]
MES MOPS TAPS CAPSO
42
6.1 7.2 8.4 9.6
Benefit of Linear pH 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
Protein Standards Using Linear pH Gradient
60.0
7.55 93
40 0
50.0
-6.
04
en -
15.9
7 - 7
2.00
-8.
53
C -
31.5
5 -9
.
30.0
40.0
banc
e [m
AU
]
ectin
-1 -
5.87
97 -
6.20
8.18
-6.
37
Tryp
sino
g
ucle
ase
A -2
2
Cyt
ochr
ome
C
10.0
20.0
Abs
orb Le
Lect
in-2
-6.
9Le
ctin
-3 -
Rib
onu C
0 5 10 15 20 25 30 35 40-5.0
0 5 10 15 20 25 30 35 40Retention Time [min]
44
Linear Correlation of Elution pH vs pI
10.510.5 10
Protein standards mAb standards
Cytochrome Cy = 1.6923x - 7.2914
R² = 0.9929
9
9.5
10 Cytochrome Cy = 1.6923x - 7.2914
R² = 0.9929
9
9.5
10 y = 1.1083x - 1.637R² = 0.9988
9
9.5
e
Ribonuclease A
8
8.5
9
red
pH
va
lue
Measured pH value
Ribonuclease A
8
8.5
9
red
pH
va
lue
Measured pH value 8
8.5
uti
on
pH
va
lue
MAb Elution pH value
L ti 3
Trypsinogen
6.5
7
7.5
Me
as
ur
Linear (Measured pH value)
L ti 3
Trypsinogen
6.5
7
7.5
Me
as
ur
Linear (Measured pH value)
7
7.5
MA
b E
lu
Linear (MAb Elution pH value)
Lectin-1Lectin-2
Lectin-3
5.5
6
7.5 8.5 9.5 10.5
Lectin-1Lectin-2
Lectin-3
5.5
6
7.5 8.5 9.5 10.56
6.5
6.5 7.5 8.5 9.5 10.5pI valuepI value MAb pI value
45
Benefit of Linear pH 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
mAb Charge Variant Separation, 0–100% B
100% B0% B
40.0 10.50
30.09.00
mAU
]
pH trace(a)
20.0
7 00
8.00
bsor
banc
e [m
10.0
6.00
7.00Ab
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)
mAb Charge Variant Separation, 0–50% B
0% B 50% B
25.0 8.50H t
20.0
mAU
]
(b) pH trace
10.07.00
bsor
banc
e [m
0.0
6.00
Ab
0 5 10 15 20 25 30 35 40-5.0 5.00
Retention Time [min]
48
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
bsor
banc
e [m
5.0
7.00
Ab
0 5 10 15 20 25 30 35 40-2.0 6.60
Retention Time [min]
49
Benefit of Linear pH 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
mAb Charge Variant Separation With Fast pH Gradient
140 11.00
(b)
0% B 100% B
100
12010.00
pH trace
(b)
809.00
ce (m
AU
)
40
60
7.00
8.00
Abs
orba
n
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
Benefit of Linear pH Gradient: High Resolution
• In most cases, we observed improved separation of the charge variants over salt gradientover salt gradient.
52
Salt vs pH Gradient 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
25.0pH gradient
53
30 min gradient, Thermo Scientific™ MabPac™ SCX-10, 10 µm, 4 × 250 mm column
Benefit of Linear pH 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
Repeat Injections of Ribonuclease A: Over 300 Runs
60 10.50
H t
Retention time reproducibility <0.8% RSD
259.00
pH trace
0
8.00
9.00
Run #300
nce
[mA
U]
50
-25
7.00
Run #200
Abs
orba
-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]
ConclusionsCh i l ti 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
Thank you
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