Stationary Phase and Mobile Phase Selection for Liquid Chromatography The presentation focuses on how to choose the appropriate mode of separation, the correct column and highlights the importance of the correct mobile phase. This approach will be applied to a wide selection of compound types ranging from proteins, peptides, glycans to small pharmaceutical molecules and their metabolites. It will also look at specific application areas for monoclonal antibody analysis, namely: titer, aggregation, charge and oxidation variant. Platform methods for biologics characterization are also discussed.

1. Stationary Phase and Mobile Phase
Selection for Liquid Chromatography
Shanhua Lin PhD
Research Scientist
Tony Edge
Scientific AdvisorScientific Advisor
October 2014

2. Introductions
Ch 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

3. 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

4. Acid / Base Equilibria
NH2 NH3
+O OH O O
-
h i li
4
www.chemicalize.org

5. Dependence of Retention Factor 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 of Selectivity 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
Bonded phase:
• 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 Silanol Groups – 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
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

14. Column Characterization (Visualization)
A C18 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 to Design 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
S ll l l tSmall molecules can enter pores,
Large molecules cannot
16

17. 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

18. 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

19. Polyethylene Oxides/Glycols
Columns: BioBasic SEC, 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 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

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 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

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 Disadvantages of 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 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
• 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 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 / MeCN
Flow rate: 0.6 mL/min
Detection: UV at 254 nm
Temperature: 30 °C
26

27. HILIC: Improved MS sensitivity 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 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 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 by Ionic 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 by pH
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 by Organic 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 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

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 Ion Pairing 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 Organic Solvent 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 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, 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 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

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 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

44. 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

45. Linear Correlation of Elution 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 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

47. 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)

48. 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

49. 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

50. 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

51. mAb Charge Variant Separation 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 Linear pH Gradient: High Resolution
• In most cases, we observed improved separation of the charge variants
over salt gradientover salt gradient.
52

53. 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
25.0
pH gradient
53
30 min gradient, Thermo Scientific™ MabPac™ SCX-10, 10 µm, 4 × 250 mm column

54. 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

55. 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]

56. Conclusions
Ch 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

57. Thank you
57