mAb Aggregates and Charge Variants...
Transcript of mAb Aggregates and Charge Variants...
Shanhua Lin, Ph.D.
mAb Aggregates and Charge Variants Analysis
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Structure of IgG and Typical Forms of Heterogeneity
2
Protein and mAb Separation by HPLC
Size Exclusion Chromatography (SEC)Size difference? MAbPac SEC-1
YES
Ion Exchange Chromatography (IEX)Charge difference?
YES
NO
MAbPac SCX-10ProPac WCX-10C ff
NO
Chromatography (IEX)
R PhNO
CX-1 pH gradient Buffer
NO
Reverse Phase Chromatography (RPC)
Hydrophobicity difference?MAbPac HIC 10
YES MAbPac RP
Hydrophobic Interaction Chromatography (HIC)
MAbPac HIC-10MAbPac HIC-20MAbPac HIC-ButylProPac HIC-10
YES
3
mAb Aggregate Analysis
4
MAbPac SEC-1
Overview• Thermo Scientific™ MAbPac™ SEC-1 is a size exclusion chromatography
(SEC) column specifically designed for separation and characterization of(SEC) column specifically designed for separation and characterization of monoclonal antibodies (mAbs).
Features• Proprietary hydrophilic bonded layer
results in minimal non-desired interactions between proteins and stationary phasestationary phase.
• Superior performance for the analysis of monoclonal antibodies, aggregates, and their fragments.
• Stable surface bonding leads to low column bleed and compatibility with MS, ELSD and Corona® CAD detection.
• Rugged reproducible column packing
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• Rugged, reproducible column packing.
Physical Data
Bonding Chemistry DiolSilica Substrate Spherical high-puritySilica Substrate Spherical, high-purity
porous silica Particle size 5 µmPore size 300 ÅPore size 300 ÅColumn housing PEEK for 4.0 mm I.D.
columnsSST for 7 8 mm and 2 1SST for 7.8 mm and 2.1 mm I.D. columns
Separation range for globular proteins
10,000 - 1,000,000g pExclusion limit for globular proteins
>1,000,000
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Column Formats Versus Target Applications
Formats Target application Why is it important7 8 × 300 mm Highest resolution separation Accurate quantitation of mAb7.8 × 300 mm Highest resolution separation
of mAb and their aggregates.Accurate quantitation of mAbaggregates. Used in the batch QCrelease assay.
4.0 × 300 mm High resolution separation of The 4.0 × 300 mm column enables4.0 300 mm4.0 × 150 mm
High resolution separation of mAb and their aggregates.
The 4.0 300 mm column enables baseline separation of mAbmonomer and dimer, required ¼ of sample comparing to the 7.8 × 300
lmm column.2.1 × 300 mm2.1 × 150 mm
Designed for SEC-MS application.
Low flow rate and low sample loading makes this format perfect for MS detectionfor MS detection.
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mAb Aggregate Analysis
mAb monomer
20 0
30.0
40.0
50.0 Column: MAbPac SEC-1, 5 µm, Dimension: a. 7.8 × 300 mm
b. 4.0 × 300 mma. 2.1 × 300 mm
Mobile phase: 50mM Sodium Phosphate pH 6.8,
mAb monomer
Aggregates
(a)
1500.0 2.5 5.0 7.5 10.0 12.5 15.0 18.0
0.0
10.0
20.0 Mobile phase: 50mM Sodium Phosphate pH 6.8,in 300 mM NaCl
Temp.: 30 ºCFlow rate: a. 760 µL/min
b. 200 µL/minc 50 µL/min
gg g
(b)
255075100125
c. 50 µL/minInj. volume: a. 10 µL
b. 5 µLa. 1 µL
Detection: 280 nmSample: mAb (1 mg/mL)
(b)
50 060.070.0
025
0.0 2.5 5.0 7.5 10.0 12.5 15.0 18.0
Sample: mAb (1 mg/mL)
(c)
0.010.020.030.040.050.0
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0.0 2.5 5.0 7.5 10.0 12.5 15.0 18.0Retention Time (min)
Impact of Ionic strength: MAbPac SEC-1
Column: MAbPac SEC-1, 5 µmFormat: 7.8 ×300 mmMobile phase: 50 mM sodium phosphate, pH 6.8
and1. 300 mM NaCl2 200 mM NaCl
200
225
250
a b c
de
2. 200 mM NaCl3. 100 mM NaCl4. 50 mM NaCl5. 25 mM NaCl6. 0 mM NaCl
Gradient: IsocraticTemperature: 30 ºC
125
150
175
6. Rs=1.64
5. Rs=1.72
Flow rate: 0.76 mL/minInj. volume: 20 µL Detection: UV (280 nm)Sample: SEC standard
a. Thyroglobulin (bovine): 0.1 mg/mLb. γ-globulin (bovine): 0.1 mg/mLc Ovalbumin (chicken): 0 1 mg/mL
50
75
100 4. Rs=1.80
3. Rs=1.90
2. Rs=2.03c. Ovalbumin (chicken): 0.1 mg/mLd. Myoglobin (horse): 0.05 mg/mLe. Vitamin B12: 0.01 mg/mL
-25
0
25
1. Rs*=2.02
MAbPac SEC-1 column has low secondary interaction its
*Rs is the resolution between γ-globulin and ovalbumin
2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0
-50
Retention Time (min)
secondary interaction, its separation is not sensitive to the mobile phase salt concentration.
9
Rs is the resolution between γ globulin and ovalbumin
Faster Aggregate Analysis
mAb monomer Column: MAbPac SEC-1 5 µm
75
100
140 mAb monomer Column: MAbPac SEC-1, 5 µm, Dimension: a. 4.0 × 300 mm
b. 4.0 × 150 mmMobile phase: 50mM Sodium Phosphate pH 6.8,
in 300 mM NaClTemp : 30 ºC
(a) 4.0 × 300 mm
25
50
75
Aggregates
Temp.: 30 ºCFlow rate: 200 µL/minInj. volume: 2 µLDetection: 280 nmSample: mAb (5 mg/mL)
-20
300
400 (b) 4.0 × 150 mm
Shorter 150 mm MAbPac SEC-1 column enables significant reduction of run times
100
200
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 25.0
-50
10
Retention Time (min)
mAb Fragments
(a)
(b)
(c)(c)
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Separation of mAb Fragments
1Column: MAbPac SEC-1, 5 µmFormat: 7.8 ×300 mmMobile phase: 50 mM sodium phosphate, 300 mM NaCl,
pH 6.8
70.01 mAb
pGradient: IsocraticTemperature: 30 ºCFlow rate: 0.76 mL/minInj. volume: 10 µL
-10.070.0
2
scFc
F(ab)2
Detection: UV (280 nm)Sample: 1. Rituximab (5 mg/mL), inject 2 µL
2. Rituximab + IdeS (2 mg/mL)3. Rituximab + Papain (2 mg/mL)4 Rit i b DTT (2 / L)
-10.070.0
3
Fc
Fab
4. Rituximab + DTT (2 mg/mL)
mAb fragments F(ab)2 and scFc, F d F b HC d LC
-10.0140
4
HC+LC
DTT
Fc and Fab, HC and LC are separated.
0.0 5.0 10.0 15.0 20.0-20
Retention Time (min)
HCLCC C
12
Retention Time (min)
Non-denaturing SEC-MS
monomer(a)Column: MAbPac SEC-1, 5 µm, Dimension: 2.1 × 150 mmMobile phase: 20 mM ammonium formateTemp.: 30 ºC
dimerFlow rate: 50 µL/minInj. volume: 1 µLDetection: Exactive Plus EMRSamples: mAb (1 mg/mL)
(b)+38
+37
+39+40
+26
(c) +27+25
+24+28
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100(a) mAb
Separation of HC and LC, Fab and Fc
50 Column: MAbPac SEC-1, 5 µm, Dimension: 4.0 × 300 mmMobile phase: 20% acetonitrile
(a)
-1070.0
Mobile phase: 20% acetonitrile,0.1% FA, 0.05% TFA
Temp.: 30 ºCFlow rate: 200 µL/minInj. volume: 5 µLD t ti 280
(b)
mAU
Detection: 280 nmSamples: (a) mAb
(b) mAb reduction by DTT(c) mAb digestion by papain
HC
LC
-10.0100
(c)Fab
50Fc
14
3.0 7.5 12.5 17.5 25.0-10
min
Denaturing SEC-MS
RT:6.88 - 16.56
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
12.71
(a)
7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5Time (min)
0
10
2010.15
8.68 10.818.89 13.7511.008.55 9.527.02 9.17 11.57 14.0114.31 15.037.41 8.117.84
Full MS of heavy chain (HC) Full MS of light chain (LC)
40
50
60
70
80
90
100
ativ
e A
bund
ance
2601.34
2341.31
2926.39
2128.55
40
50
60
70
80
90
100
lativ
e A
bund
ance
2109.962201.64
1947.72 2301.67
2531.731875.652411.25
2664.912812.902978.34
3164.421808.68
3375.33
2323.12
3006 032839.03
Full MS of heavy chain (HC) Full MS of light chain (LC)(b) (d)
+10+9
+8+11
+12
+20+18 +16
1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z
0
10
20
30
40
Rel
a
1951.241801.26
1672.693344.411561.09 2619.36 2946.82
3901.713121.58 3601.602385.901463.66 2169.161988.65 3367.772754.361322.721101.60
1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z
0
10
20
30
Rel
1746.42
1688.25 3616.421557.72
3006.03
3193.701110.59 1340.65 3406.59
3894.423345.21
( ) ( )
12
+7+13
+14
G0
G1G0+Lys
G1+LysG2
(c) (e)
15
G2
Chromatographic Conditions for MAbPac SEC-1
Column ID (mm) 2.1 4.0 7.8
Flow rate (µL/min) 50-75 200-300 760-1 000Flow rate (µL/min) 50-75 200-300 760-1,000
UV flow cell Micro (180 nL) Semi-micro(2.5 µL)
Analytical (11 µL)
Tubing ID (µm) 50-75 100 150-250Tubing ID (µm) 50 75 100 150 250
Sample Loop Size (Pull Loop WPS (µL))
1 5 20
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Tips and Tricks for SEC Analysis
• If you are getting low resolution separation, check:• System setup: connection tubing, flow rate, flow cell size, injection method.• Use a protein mix standard to QC the system: Bio-rad Gel Filtration Standard p y
#151-1901
160 tB12
75
100
125
mAU
Column: MAbPac SEC-1, 5 µm, Dimension: 4 × 300 mmMobile phase: 0.3 M NaCl in 50mM Sodium Phosphate pH 6.8Temp.: 25 ºCFlow rate: 200 µL/minInj. volume: 5 µLDetection: 280 nmSample: MAb (5 mg/mL)
Thyr
oglo
bulin
gam
ma-
blob
ulin
Ova
lbum
in.
Myo
glob
in
Vi t
0.0 5.0 10.0 15.0 20.0 25.0-10
25
50
min
• If you observe high pressure, check to make sure that the flow cell is not blocked.
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mAb Charge Variant Analysis
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Charge Variant Analysis
Untreated mAb
Lysine VariantsYYK
YYProPac WCX-10 Column
10 µm Nonporous Polymeric Beads
Polymeric graftsWCX SCX WAX
YYKK
K
Acidic VariantsBasic Variants
WCX, SCX, WAX,SAX
Boundary(Cross-linked YYCarboxypeptidase B (hydrophilic layer)Core(Highly cross-linkedEVB-DVB)
digested mAb
Acidic VariantsBasic Variants
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Gold Standard for mAb Analysis
P P WCX 10 C lProPac WCX-10 Column
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Next-generation CEX Column
YYYYLysine Variants
YYKK
YYK
YY
Acidic YY
YYMAbPacSCX-10 Column
VariantsBasic Variants
YYKK
KLysine Variants
ProPac WCX-10 column, 4×250 mm
Acidic
KK
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MAbPac SCX-10 column, 4×150Variants
Basic Variants
4×150 mm 15 min. total analysis time
mA
U
MAbPac SCX-10 column, 4×250 mm60 min. total analysis time
0 10
0
5 15Minutes
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Improve resolution or sample throughput through column chemistry
Thermo Scientific CX-1 pH Gradient Buffers
Buffer A Buffer B
pH 5.6 10.2
Form Liquid Liquid
• Dilute buffers 10-fold with DI water
Form Liquid Liquid
Concentrate 10X 10X
Shi i R R• A linear pH gradient (pH 5.6 - 10.2)
is generated by running a linear pump gradient from 100% Buffer A
Shipping condition
Room Temp
Room Temp
Storage4 ~ 8 C 4 ~ 8 Cp p g
to 100% Buffer B • Generic, fast & high-resolution!
condition4 ~ 8 C 4 ~ 8 C
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pH gradient platform method for charge variant analysis
Protein and MAb Separation on IEX Columns
Salt Gradient pH Gradient
• Most widely used method • Can predict elution profileMost widely used method
• Relatively simple to make the buffer
Can predict elution profile with pI value
• Lower salt concentration in
• Takes longer to optimize the separation condition (pH,
collected fractions
• In many cases, improved salt concentration) resolution was observed
• Difficult to generate a linear H di tpH gradient
23
pH Gradient Buffers – How Do They Work?
P i El i M h i IEX
+IsoelectricPoint (pI)
COO -
Protein Elution Mechanisms on IEX
Buffer pH typically < pI
Cation-ExchangeChromatography
COOH
NH3R +
Cationic protein binds to
negatively charged cation exchanger
+ ++
++
+ ++
05 6 7 8 9 10 11 12
Buffer/System pHNH3
R +COOH
4
Buffer pH typically > pI
Anion-ExchangeChromatography
COO -
Anionic proteinbinds to
positively chargedanion exchanger
- -- -
-
- - -
pH range covered by CX-1 pH gradient buffers
–Protein net charge vs. pH
NH2R
COO
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Linear pH Gradient by Zwitterionic Buffer Cocktail
10.510.5
y = 0.1577x + 4.9755R² = 0.9996
9.5
valu
e
y = 0.1577x + 4.9755R² = 0.9996
9.5
valu
e
7.5
8.5
easu
red
pH v
Measured ValueLinear (Measured Value)7.5
8.5
easu
red
pH v
Measured ValueLinear (Measured Value)
6.5
Me
6.5
Me
5.50 10 20 30 40
Retention Time [min]
5.50 10 20 30 40
Retention Time [min]
25
Protein Standard – CX-1 pH Gradient Buffer
90 11
MAbPac SCX-10 Column, 4×250 mmPeak label: protein name – elution pH
pH trace60
10
Lectin-1 - 6.11
p ace
40
60
8
9
Cytochrome C - 10.15
20
6
7Lectin-2 - 6.28
Lectin-3 - 6.45 Trypsinogen - 7.75Ribonuclease A - 8.72
0 5 10 15 20 25 30 35 40-10 5
0
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Protein Standard – Phosphate-based pH Gradient
70 10.0pHMAbPac SCX-10 Column, 4×250 mm
50
60
8 5
9.0
9.5
pH trace30.
40
7.5
8.0
8.5
10
20
6.5
7.0
2
0 5 10 15 20 25 30 35 40-10
0
5.5
6.012
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Why pH Gradient Buffers?
• Traditional salt gradient using ion exchange chromatography
• Needs to be tailored for individual charge 10.0
15.0
30.0
gvariants
0 0
5.0 Salt gradient
• Proprietary pH buffer formulations
• Fast robust and reproducible pH gradients
0.0 5.0 10.0 15.0 20.0 25.0 30.0
0.0
min
%B: 10.0
15.0
50.0
• Fast, robust and reproducible pH gradients
• Ready to use with existing CX columns and systems 5.0
10.0
pH gradient
• Simple to optimize and easily automated
• Applicable to the majority of MAbs0.0 5.0 10.0 15.0 20.0 25.0 30.0
0.0
min
%B: 25.0
25.0gradient
28
• Applicable to the majority of MAbs
pH Gradient Buffers Product Description – Introduction
Buffer A Buffer B
pH 5.6 10.2
F Li id Li idForm Liquid Liquid
Concentrate 10X 10X
• Simple to use• Dilute buffers 1:10 in DI water• A linear pH gradient (pH 5 6 - 10 2) Shipping
conditionRoom Temp
Room Temp
Storage
• A linear pH gradient (pH 5.6 - 10.2) is generated by running a linear pump gradient from 100% eluent A to 100% B. g
condition 4 – 8 C 4 – 8 C• It is generic, fast, and high-res!
29
Benefit of Linear pH Gradient: Compatible with WCX and SCX
• pH gradient can be used with both weak cation exchange (ProPac WCX-10) d t ti h l (MAbP SCX 10)10) and strong cation exchange columns (MAbPac SCX-10)
• There is a pH gradient delay with the weak cation exchange column.
30
ProPac WCX-10 vs MAbPac SCX-10
250 10.50
ProPac WCX 10 4 × 250 mm
100 8.00
U)
ProPac WCX-10, 4 × 250 mm
350 10.50
-50 5.00
banc
e (m
AU
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
125
250
8.00Abs
orb MAbPac SCX-10, 4 × 250 mm
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0-50 5.00
R t ti Ti ( i )
31
Retention Time (min)
Benefit of Linear pH Gradient: Generic Approach
• A generic approach to charge variant analysis, covering the pH range 5.6 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 curve
32
Protein Standards Using Linear pH Gradient
0 0
60.0
7 -7
.55
3 -9.9
3
40.0
50.0
mAU
]
-5.8
7 -6
.04
0 6.37 sino
gen
-15.
9 7
A -2
2.00
-8.
5
rom
e C
-31
.55
20.0
30.0
Abs
orba
nce
[m
Lect
in-1
-n-
2 -6
.97
-6.2
ctin
-3 -
8.18
-6
Tryp
Rib
onuc
leas
e
Cyt
ochr
10.0 Lect
inLe
c
0 5 10 15 20 25 30 35 40-5.0
Retention Time [min]
33
Linear Correlation of Elution pH vs pI
Cytochrome Cy = 1.6923x - 7.2914
R² = 0.992910
10.5
Cytochrome Cy = 1.6923x - 7.2914
R² = 0.992910
10.5
Ribonuclease A
R 0.9929
8 5
9
9.5
valu
e
Ribonuclease A
R 0.9929
8 5
9
9.5
valu
e
Trypsinogen
Ribonuclease A
7.5
8
8.5
Mea
sure
d pH
v
Measured pH value
Linear (Measured pH value)
Trypsinogen
Ribonuclease A
7.5
8
8.5
Mea
sure
d pH
v
Measured pH value
Linear (Measured pH value)
Lectin-1Lectin-2
Lectin-3
6
6.5
7M )
Lectin-1Lectin-2
Lectin-3
6
6.5
7M )
5.57.5 8.5 9.5 10.5
pI value
5.57.5 8.5 9.5 10.5
pI value
34
mAb Standards Using Linear pH Gradient
160
180
200
9 50
10.00
10.50
pH trace
120
140
160
8.50
9.00
9.50
U)
60
80
100
7 00
7.50
8.00
sorb
ance
(mAU
20
40
60
6.00
6.50
7.00
Abs
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0-20
0
5.00
5.50
35
Retention Time (min)
Linear Correlation of Elution pH vs pI
y = 1.1083x - 1.637R² = 0.9988
9.5
10
8.5
9
pH v
alue
7.5
8
Ab E
lutio
n p
MAb Elution pH value
Linear (MAb Elution pH value)
6.5
7
MA
66.5 7.5 8.5 9.5 10.5
MAb pI value
36
Top-selling mAbs Analyzed by pH Gradient Method
25 0
37.5
50.0
70.0
Rituxan2
25 0
37.5
50.0
70.0
Herceptin
15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0-10.0
0.0
12.5
25.0
Retention Time (min)
1 3
15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0
-10.0
0.0
12.5
25.0
Retention Time (min)
15.0
18.0
Humira15 0
20.0
Avastin
5.0
10.0
5.0
10.0
15.0
15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0
-2.0
Retention Time (min)
15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0
-2.0
Retention Time (min)
37
Platform method for mAb charge variant analysis
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• By running a shallower pH gradient a higher resolution separation is obtained (e.g. 50-100%, rather than 0-100%B)
38
mAb Charge Variant Separation, 0–100% B
100% B0% B
40.0 10.50pH trace(a)
20.0
30.0
8.00
9.00
ance
[mA
U]
10.0
6.00
7.00Abs
orba
0 5 10 15 20 25 30 35 40-5.0 5.00
Retention Time [min]
39
*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
20 0
25.0 8.50(b) pH trace
10 0
20.0
7.00
ance
[mA
U]
0 0
10.0
6.00
Abs
orba
0 5 10 15 20 25 30 35 40-5.0
0.0
5.00
Retention Time [min]
40
mAb Charge Variant Separation, 25–50% B
25% B 50% B
16.0 8.00(c) pH trace
10.0 7.50
7.75
ance
[mA
U]
5.0
7.00
7.25
Abs
orba
0 5 10 15 20 25 30 35 40-2.0 6.60
Retention Time [min]
41
Benefit of Linear pH Gradient: Fast Analysis
• By using• A smaller particle (5 µm rather than 10 µm)
• A shorter cation-exchange column (4 × 50 mm)
MAb charge variant profile can be quickly determined within 20 minMAb charge variant profile can be quickly determined within 20 min.
42
mAb Charge Variant Separation With Fast pH Gradient
250 10.50
0% B 100% B
150
200
9.00
U)
pH trace
100
7 00
8.00
bsor
banc
e (m
A
0
50
6.00
7.00Ab
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0-50 5.00
Retention Time (min)
43
Ultra Fast pH Gradient: 10-Minute Run
140 11.00
0% B 100% B
100
120
9.00
10.00
U)
pH trace
60
80
8.00
bsor
banc
e (m
A
20
40
6.00
7.00
1
Ab
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0-20
0
5.00
1
Retention Time (min)
44
Retention Time (min)
Fast Method Development
5 min gradient
10 min gradient
3 steps method development1
0.8 min gradient1. 10 minutes 0100 % B in 10 minutes2. 2040 %B in 5 minutes3. 1827 %B in 0.8 minutes
Number of charge variants and resolution maintained for sub-
45
gminute gradient
Benefit of Linear pH Gradient: High Resolution
• In most cases, we observed improved separation of the charge variants over salt gradient.
46
Salt vs pH Gradient IEC of mAb Sample
15 0
10.0
15.0
30.0
5.0
Salt gradient
0.0 5.0 10.0 15.0 20.0 25.0 30.0
0.0
min
%B: 10.0
g
15.0
50 0
10.0
50.0
0.0
5.0
i
%B: 25.0
25.0pH gradient
47
30 min gradient, Thermo Scientific™ MabPac™ SCX-10, 10 µm, 4 × 250 mm column0.0 5.0 10.0 15.0 20.0 25.0 30.0 min
Benefit of Linear pH Gradient: Great Precision
• The retention times in pH gradient IEC are highly reproducible
• This makes prediction of pI very consistent
48
60 10.50
Repeat Injections of Ribonuclease A: Over 300 Runs
259 00
pH trace
0
8.00
9.00
Run #300
ance
[mA
U]
-50
-25
7.00
Run #200
Abs
orba
-756.00
Run #100
Run #5
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0-100 5.00
Run #5
Retention Time [min]
Retention time reproducibility <0 8% RSD
49
Retention time reproducibility <0.8% RSD
CX-1 pH Gradient Buffer Kit (10X): Simple To Use
110
Lot 1 (130523 / 130528)Lot 2 (130524 / 130529)Lot 3 (130531 / 130530)
60
80
mA
U)
Lot 3 (130531 / 130530)
40
60
bsor
banc
e (m
20
Ab
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0-10
50
Retention Time (min)
Tips and Tricks for IEC Analysis
• Eluents for ProPac WCX-10 and MAbPac SCX-10 columns should always contain small amount of electrolytes. Never wash the columns with DI water.
• Try to avoid using organic solvent in the mobile phase. If organic solvent is i d d k th i t t i t t!required, do keep the organic content consistent!
• Use inert HPLC system to avoid metal contamination.• If you are want to try out pH gradient method, start with MAbPac SCX-10, 10
4 ×250 lµm, 4 ×250 mm column. • Run a protein standard mix (Lentil Lectin, Ribo A, Trypsinogen, and CytC) to
make sure the system is working properly. Monitoring the pH and Conductivity will be a great PLUS!will be a great PLUS!
• To speed up your analysis, use either shorter format (such as MAbPac SCX-10, 10 µm, 4 ×150 mm column) or smaller particle (such as 5 µm).
• If your sample do not bind and elute immediately check protein pI and pH of theIf your sample do not bind and elute immediately, check protein pI and pH of the sample buffer.
51
Thank you!
52