Poster_RecoveryXV (5Cols)_D2_Mise en page 1 26/07/2012 15:57 Page 1
Characterization of Interaction Mechanisms on Mixed-Mode Chromatography Sorbents:
From Separation Optimization to HCPs Identification for Better Exploitation in MAb Purification
Jérôme Pezzini 1,2, Xavier Santarelli 1, Magali Toueille 2, René Gantier 2 ( 1Ecole Nationale Supérieure de Technologie des Biomolécules de Bordeaux, Bordeaux, France 2Pall Life Sciences, Cergy, France)
Table 1
Potential Protein/Ligand Interaction Using MEP, HEA and PPA HyperCel Sorbents
The resin structures are presented in Figure 1. Mixed-mode chromatography exploits
multiple, distinct protein-ligand interactions to adsorb target proteins or impurities (see
Table 1).
p-p Interaction
Thiophilic Interaction
MEP HyperCel
Ethyl group
Amine (pKa = 4.8)
Pyridine group
Thio-ether group
HEA HyperCel
Hexyl group
Amine (pKa ~ 8)
—
—
PPA HyperCel
Propyl group
Amine (pKa ~ 8)
Phenyl group
—
Figure 3
Contour Plots Obtained for the Optimization of HCP Removal and Yield of Recovery for MAb
Capture Step Using MEP, HEA and PPA HyperCel Sorbents
OPTIMIZATION OF CAPTURE STEP FOR THE PURIFICATION OF
A MONOCLONAL ANTIBODY USING MIXED-MODE SORBENTS
A: Result on MEP HyperCel Sorbent
Figure 2
Procedure to Screen pH and Conductivity Conditions to be Used on MEP, HEA and PPA
HyperCel Sorbents to Optimize the MAb Yield of Recovery and Purity at Capture Step of the
Purification Process
Elution pH
1. Design of Experiment (DoE)
• Critical parameters (pH/conductivity for wash and elution)
• Quality attributes (yield of recovery, HCP removal)
Uncharged at
near-neutral pH
CH2 CH2 CH2 S
ad
< 4400
4400 – 4500
4500 – 4600
4600 – 4700
4700 – 4800
> 4800
2. Screening on 96-Well Plates
• AcroPrep™ Advance Filter Plates
• Vacuum manifold
Optimum conditions:
u Wash pH 5.5, conductivity 7 mS/cm
u Elution pH 4.0, conductivity 3 mS/cm
Low pH wash results in:
u Desorption of basic proteins
(charge repulsion)
u Partial loss of MAb (weak
hydrophobic binding)
0.006
Optimum conditions:
u Wash pH 6.5, conductivity 15 mS/cm
u Elution pH 4.0, conductivity 3 mS/cm
0.005
0.004
0.003
0.002
0.001
Hold Values
Elution Cond. 3
Elution pH
4
6.75
5.50
5
10
15
20
5
Wash Conductivity (mS/cm)
5.0
10
15
20
Wash Conductivity (mS/cm)
HCP Content (ng/mL) vs.
MAb Yield (%) vs.
Elution pH and Elution Conductivity Elution pH and Elution Conductivity
HCP Conc. (ng/mL)
MAb Yield (%)
< 2400
2400 – 3000
3000 – 3600
3600 – 4200
4200 – 4800
> 4800
< 15
15 – 25
25 – 35
35 – 45
45 – 55
> 55
Hold Values
Wash Cond. 7
Wash pH 5.5
4.5
4.0
86
1500
HEA HyperCel
90
730
10
15
20
5
Min
11
15
20
< 1550
1550 – 1600
1600 – 1650
1650 – 1700
> 1700
Hold Values
Elution Cond. 3
Elution pH
4
MAb Yield (%) vs.
Wash pH and Wash Conductivity
MAb Yield (%)
< 50
50 – 55
55 – 60
60 – 65
> 65
7.00
6.75
6.50
Hold Values
Elution Cond. 3.2
Elution pH
4
6.25
6.00
5.75
5.50
5
10
15
20
5
Wash Conductivity (mS/cm)
5.0
12
13
10
15
20
Wash Conductivity (mS/cm)
HCP Content (ng/mL) vs.
MAb Yield (%) vs.
Elution pH and Elution Conductivity Elution pH and Elution Conductivity
HCP Conc. (ng/mL)
MAb Yield (%)
< 2500
2500 – 3000
3000 – 3500
3500 – 4000
4000 – 4500
> 4500
< 30
30 – 40
40 – 50
50 – 60
60 – 70
> 70
Hold Values
Wash Cond. 7
Wash pH 5.5
4.5
4.0
Hold Values
Wash Cond. 15
Wash pH 6.5
3.5
3.0
10
15
20
5
Elution Conductivity (mS/cm)
10
10
7.25
HCP Conc. (ng/mL)
10
15
20
Elution Conductivity (mS/cm)
14
CH2 CH2
C: Result on PPA HyperCel Sorbent
Elution pH
CH2 (CH2)4 CH3
4. Result Analysis
• Sweet spot for optimum performances
250-300
7.5
200-250
7.0
6.5
150-200
6.0
5.5
< 700
700 – 800
800 – 900
900 – 1000
1000 – 1100
> 1100
Hold Values
Elution Cond. 3
Elution pH 3.8
Positive charge can contribute
to binding of acidic proteins or
inhibits binding of basic proteins.
It can moderate attractive or
repulsive interactions based
on pH and conductivity.
CH2 CH2
MAb Yield (%)
< 40
40 – 50
50 – 60
60 – 70
70 – 80
> 80
7.00
6.75
6.50
Hold Values
Elution Cond. 3
Elution pH 3.8
6.25
6.00
4.0
4.2
4.4
4.6
Load pH
5. Transfer to Column
MAb Yield (%) vs.
Wash pH and Wash Conductivity
7.25
Wash pH
Low pH + high conductivity wash allow:
u Desorption of basic proteins
(charge repulsion)
u No loss of MAb (hydrophobic binding)
300-350
HCP Content (ng/mL) vs.
Wash pH and Wash Conductivity
HCP Conc. (ng/mL)
4.8
5.0
Low pH + low conductivity elution allow
desorption of:
u Acidic proteins (charge repulsion)
u Hydrophobic proteins including MAb
(weak hydrophobic binding)
Optimum conditions:
u Wash pH 5.5, conductivity 23 mS/cm
u Elution pH 3.8, conductivity 3 mS/cm
5.75
5.50
5
10
15
20
5
Wash Conductivity (mS/cm)
10
15
20
Wash Conductivity (mS/cm)
HCP Content (ng/mL) vs.
MAb Yield (%) vs.
Elution pH and Elution Conductivity Elution pH and Elution Conductivity
5.0
HCP Conc. (ng/mL)
MAb Yield (%)
< 50
50 – 500
500 – 1000
1000 – 1500
> 1500
< 10
10 – 20
20 – 40
40 – 60
60 – 80
> 80
Hold Values
Wash Cond. 23
Wash pH
5.5
4.5
4.0
Hold Values
Wash Cond. 23
Wash pH 5.5
3.5
pKa ~ 8
4000
4000
3000
3000
3000
2000
2000
2000
1000
1000
0
0
5
6
40
7
60
8
9
100
80
20
MW (kDa)
1000
0
0
5
6
pI
40
7
60
8
9
100
80
20
MW (kDa)
0
0
5
6
pI
40
7
60
8
9
100
80
20
MW (kDa)
u
Less HCPs identified in elution fraction from HEA and PPA HyperCel sorbents
compared to MEP HyperCel sorbent (PPA HyperCel sorbent is the most selective).
u
Most acidic (pI < 5) and highest molecular weight (> 50 kDa) HCPs are removed using
mixed-mode sorbents (eluted at lower pH than MAb):
– Very low pH is required to induce charge repulsion for acidic proteins
– More chance of presence of hydrophobic patches on high molecular weight proteins,
facilitating stronger interactions
u
PPA HyperCel sorbent efficiently removed proteins with pI < 5.5 due to higher
ligand hydrophobicity.
u
More basic proteins (pI > 7) are co-eluted with MAb from MEP HyperCel sorbent.
u
A combination of similar pI and hydrophocity than that of MAb is required for HCPs to
co-elute with the MAb.
IDENTIFICATION OF HCP CO-ELUTING WITH MAb
A clarified CHO CCS without MAb expressed was loaded on the three mixed-mode
sorbents and the optimum wash and elution conditions were applied. Trypsin digested
elution fractions were analyzed by online capillary HPLC to identify and quantify (relative
abundance) HCPs present. The most abundant HCPs identified in elution fractions are
presented in Table 3. The HCP breakdown in pI and MW in each elution fraction is shown
in Figure 4.
Identity of HCP
Ubiquitin
SH3 BGRL
Beta-2-microglobulin
Galectin-1
Stathmin
Peptidyl-prolyl cis-trans isomerase A
Thioredoxin
Cofilin-1
Peptidyl-prolyl cis-trans isomerase B
Metalloproteinase inhibitor 2
Peroxiredoxin-1
Glutathione S-transferase PI
Cathepsin L1
Cathepsin Z
HnRNPA2B1
Nuclear migration protein nudC
Annexin A2
Annexin A1
Fructose-bisphosphate aldolase a
Macrophage-capping protein
Actin, cytoplasmic 2
Alpha-enolase
Serine protease HTRA1
Elongation factor 1-alpha-1(III) chain
Lysosomal protective protein
Pyruvate kinase isozyme M1/M2
Dystroglycan
Transketolase
78 kDa glucose-regulated protein
Heat shock cognate 71 kDa protein
Prelamin-A/C
Elongation factor 2
Collagen alpha-1(III) chain
Vinculin
HSPG
< 100 ng
3.0
5
10
15
Elution Conductivity (mS/cm)
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Web: www.pall.com/biopharm
4000
460
Table 3
HCP Identity, Molecular Weight (MW), Isoelectric Point (pI) and Mass Detected in Elution
Fraction from HEA, PPA and MEP HyperCel Sorbents
Elution Conductivity (mS/cm)
HCP Content (ng/mL) vs.
Wash pH and Wash Conductivity
5
9
93
HCP
(ng)
Hold Values
Wash Cond. 7
Wash pH 5.5
3.5
ELution pH
(Spacer)
MEP HyperCel
HCP
(ng)
6.25
5.75
5.0
PPA HyperCel
Hold Values
Elution Cond. 3
Elution pH
4
6.50
0
8
H+
H+
N CH2
HCP Content (ng/mL)
6.00
Moderate acidic pH + low conductivity
elution allow desorption of:
u Acidic proteins (charge repulsion)
u Hydrophobic proteins (weak
hydrophobic binding)
Moderate acidic pH + low conductivity
elution allow desorption of:
u Acidic proteins (charge repulsion)
u Hydrophobic proteins (weak
hydrophobic binding)
HCP (ppm)
pKa ~ 8
MAb Yield of Recovery (%)
< 40
40 – 50
50 – 60
> 60
7.00
B: Result on HEA HyperCel Sorbent
Filter plate
8.0
N
Sorbent
PPA HyperCel
HCP
(ng)
pI
pH
Sorbent bed
8.5
(Spacer)
Table 2
On-Column Experiments for the Monoclonal Antibody Yield and HCP Content
HEA HyperCel
MEP HyperCel
MAb Yield (%)
Elution Conductivity (mS/cm)
Buffer
3. Analytical Testing
• Aggregate quantification (SEC HPLC)
• HCP quantification (ELISA)
• MAb elution yield (OD280nm)
7.25
HCP Conc. (ng/mL)
5
pKa = 4.8
HEA HyperCel
Transfer of optimum conditions (wash and elution) performed on prepacked columns
(5 mL, 0.5 cm I.D., 8 cm bed height, 2.5 minutes residence time) for the three mixed-mode
sorbents (see Table 2).
Figure 4
HCP Mass Versus pI and Molecular Weight Measured in Elution Fraction from MEP, HEA and
PPA HyperCeI Sorbents
MAb Yield (%) vs.
Wash pH and Wash Conductivity
3.0
Lo
AU
Figure 1
Ligand Structure of MEP, HEA and PPA HyperCel Mixed-Mode Sorbents
Low conductivity wash allows:
u Desorption of hydrophilic proteins
(weak hydrophobic binding)
u No loss of MAb (hydrophobic binding)
HCP Content (ng/mL) vs.
Wash pH and Wash Conductivity
Wash pH
A clarified CHO CCS containing a MAb (isoelectric point pI ~7) was used as sample load.
The feedstock contained 2.3 mg/mL MAb and 92 µg/mL or 40,000 ppm (ng/mg MAb) host
cell proteins. The procedure used to optimize the capture step is detailed in Figure 2.
and offers alternative/complement to conventional hydrophobic interaction (HIC)
or hydroxyapatite.
O
Transfer on Column of Optimized Conditions
PPA HyperCel
Mixed-mode chromatography offers new solutions to separations where:
u Traditional chromatographic methods (ion exchange, HIC, affinity…) are not effective,
u Feedstream conductivity is too high for efficient capture on traditional ion
exchange resins,
u Affinity ligands are too expensive,
MEP HyperCel
Optimum pH and conductivity for wash and elution steps on MEP, HEA and PPA HyperCel
sorbents were identified using high throughput experiment (HTE) in 96-well plates.
The obtained contour plots for both wash and elution optimization are presented in Figure 3.
ELution pH
MIXED-MODE CHROMATOGRAPHY:
MEP, HEA AND PPA HYPERCEL SORBENTS
Ionic Interaction
Wash pH
We have evaluated some of the fundamental mechanisms that rule protein interaction
on three mixed-mode sorbents (MEP, HEA and PPA HyperCel™ sorbents) and studied
their performance for MAb purification. Using design of experiments and high throughput
96-well plates, the binding characteristics of CHO host cell proteins (HCP) from a cell
culture supernatant (CCS) were evaluated. Subsequently, mass spectrometry was utilized
to identify HCPs co-eluting with the MAb under optimized purification conditions.
The analysis of the HCPs physico-chemical properties gave a deeper insight, for each
sorbent, of the mechanism controlling the separation between the target MAb and
contaminant proteins.
Hydrophobic
ELution pH
Mixed-mode (or multi-mode) chromatography is now implemented in many processes
for the purification of monoclonal antibodies (MAbs) as an alternative to hydrophobic
interaction chromatography (HIC) or ion-exchange chromatography (IEX). The number of
commercially available mixed-mode sorbents is expanding, with each ligand presenting
different combinations of hydrophilic, electrostatic and hydrophobic moieties for protein
interaction. A deeper understanding of the mixed-mode binding mechanism, as distinct
from IEX or HIC, is therefore critical to optimize the use of the technique for MAb purification.
Sorbent
Elution Cond
INTRODUCTION
High Throughput Experiment for the Determination of Optimum
Wash and Elution Conditions
20
5
10
15
Elution Conductivity (mS/cm)
100 – 249 ng
MW (kDa)
8.6
12.8
13.8
14.7
17.1
17.8
18.0
18.4
20.2
21.7
22.3
23.4
24.1
27.0
37.4
38.4
38.5
38.6
39.2
39.2
41.8
47.0
48.9
50.1
51.4
57.7
67.6
67.6
70.5
70.7
74.2
95.2
95.5
116.6
396.1
pI
6.50
4.87
6.89
5.53
5.76
7.88
6.94
8.26
9.42
6.47
6.50
6.89
5.23
5.87
8.97
5.17
7.53
7.15
8.40
6.73
5.31
6.36
6.73
9.10
5.55
7.42
9.41
7.23
5.01
5.37
6.54
6.23
9.37
5.77
5.85
250 – 749 ng
HCP in
HEA Elution
ND
ND
ND
ND
ND
ND
ND
ND
ND
HCP in
PPA Elution
HCP in
MEP Elution
ND
ND
ND
ND
CONCLUSION
ND
ND
ND
ND
ND
Mixed-mode chromatography on MEP, HEA and PPA HyperCel sorbents are
efficient tools for MAb purification:
u
Optimization of separation conditions using a high throughput experiment
can be used to better understand interaction mechanisms on each sorbent.
u
The identification of HCP co-eluting with the MAb reinforced the understanding of separation mechanisms.
u
The three mixed-mode sorbents are complementing each other to achieve
good MAb purification.
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
750 – 3500 ng
> 3500 ng
20
ND = Not Determined
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