Chromatography: Chromatographic strategies for IVIG purification – Part 2

Merck KGaA
Darmstadt, Germany
Chromatographic
strategies for IVIG
purification
Thierry Burnouf PhD.
Vice-Dean, College of Biomedical Engineering, Taipei Medical
University
Josephine Cheng
Associate Director Plasma Segment APAC, Process Solutions,
Life Science
– Part 2

The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.
Chromatographic Strategies for IVIG Purification - Part 2 | 29.11.182

01 Introduction
02 Case Study Strategies
03 Results
04 Conclusion

Immunoglobulin G (IgG,
intravenous and
subcutaneous)
 Important plasma-derived product
used to treat patients with various
immunologic, neurologic, and
hematologic conditions, e.g. PIDD,
ITP, CIDP, leukemia, Kawasaki
Syndrome
 Market growth driven by increasing
use of immunoglobulins in various
therapeutic areas, aging population,
and increasing plasma collection

Therapeutic IgG
production
 Quality criteria including low
residual level of contamination by
other proteins such as IgA, IgM,
proteolytic enzymes, or Factor
XI/XIa
 Increasing use of chromatography
as an essential step for fine-tuning
purification of IgG and removal of
protein contaminants

Product Characteristics
Base Material Polymethylacrylate polymer
Mean Particle Size 40-90 μm
Functional Group
Strong anion exchanger
trimethylammoniumethyl
Surface Chemistry
Tentacular ligand chemistry
composed of positively-
charged groups
Benefits
Efficient separation and a
high binding capacity even
at high flow rates, long
product lifetime
A reliable anion-exchange chromatography resin to purify
plasma-derived IgG
Fractogel® EMD TMAE (M) Resin
Tentacular ligand chemistry
6Chromatographic Strategies for IVIG Purification - Part 2 | 29.11.186

Polish
+
+
+
+
+
+
-
+
++
+
-
-
-
LOAD
Load:
•IgG Feed
Flow-through
•IgG
Retention
•IgA, IgM, proteolytic
enzymes, Factor IX,
Factor IXa, and other
contaminants
Negative Mode Anion-Exchange Chromatography
Contaminants are bound onto the column, IgG in the flow-through
Fractogel®
EMD TMAE (M)
Resin
Purified IgG
IgG Feed

Design | Methods | Results
Case
Study

Case Study
One step IVIG purification by AEX chromatography
Phase 1a
Optimization of
chromatographic
conditions
Phase 2
Tests of resin stability
and process
performance up to
200 cycles, including
cleaning and
sanitization
Phase 1b
Analysis of IgG purity
and reproducibility
using optimized
conditions
Experimental design

Phase 1a
Optimization of
chromatographic
conditions
Fractogel®
EMD TMAE
(M) Resin
IgG Feed
Purified IgG
IgA/IgM Capture

 Representing worst
case scenario of
precipitate (I)+II+III
 Caprylic Acid treated
human plasma derived
IgG: CA-IgG
Phase 1a
Preparation of IgG fraction:
Cryoprecipitation
Caprylic Acid Treatment
Chromatography
1
2
3
4
Fractogel® EMD
TMAE (M) Resin
TFF Dialysis
Cogent® μScale TFF System

 pH
 Conductivity
 Total protein (Biuret)
 Albumin (bromocresol green)
 IgG (ELISA and immunonephelometry)
 IgA (ELISA)
 IgM (ELISA)
 IgG subclasses (ELISA)
Analytical methods used
 Gamma protein determination by zone
electrophoresis
 SDS-PAGE reducing/non-reducing conditions
 Thrombogenic and proteolytic activity potential
 FXIa assay (FXIa Hyphen)
 Thrombin generation assay (Technoclone)

Phase 1a
IgG feed purity check: SDS-PAGE
←IgG
13
←Heavy chain
←Light chain
SDS-PAGE
• Under non-reduced conditions:
one main protein band of ca.
160kDa (intact IgG)
• Under reduced conditions:
protein bands of ca. 50kDa (IgG heavy
chain) and ca. 25kDa (IgG light chain)
Non-reduced Reduced

ELISA confirms:
• Higher content in IgA and IgM in the CA-IgG (compared to a control IgG prepared from fraction II)
• CA-IgG is roughly equivalent to a Cohn precipitate (I)+II+III (worst case fraction)
• CA-IgG fraction contains a high, physiological proportion of IgA and IgM (worst case fraction)
Phase 1a
IgG, IgA and IgM mean content in “CA” feed by ELISA
C A -Ig G
8 0 .1 4 % Ig G
1 7 .7 2 % Ig A
2 .1 4 % Ig M
C o n tro l-Ig G F e e d
9 8 .7 6 % Ig G
1 .2 2 % Ig A
0 .0 2 % Ig M

Chromatographic steps Typical loading chromatographic conditions
 1 mL packed column (convenient for quick
investigations)
 Expected loading capacity: 150-200 mg of
(purified) IgG per 1 mL of resin
Phase 1a
Chromatographic conditions testing: pH 5.7, pH 6.0 and pH 6.3
Parameter Parameter values
Residence time 6.5 min
Linear flow rate 185 cm/hr
Pre-equilibration
250 mM sodium acetate
buffer, 3 CV
Equilibration
buffer, 5 CV
Wash
buffer, 3 CV
Regeneration
1.5 M NaCl in 250 mM
sodium acetate, pH 4.5, 2
CV
CIP
0.5 M/1.0 M NaOH, 3 CV;
10 min. residence time
(total contact time: 30 min.)
0
1000
2000
mL
mAu
80604020
IgG flow-through
IgA and IgM waste

Phase 1a Result
pH 5.7 to pH 6.3 range has limited impact on chromatographic
performance
Mean of 3 cycles at each pH
10 15 20 25 30 35 40
0
200
400
600
800
1000
IgG injection (mL)
IgG(mg/dl)
pH5.7
pH6.0
pH6.3Feed
Content of IgG in flow-through
Wash
• Limited pH impact
• Gel robustness
Limited loss of IgG

Phase 1a Result
Impact of pH on IgA chromatographic behavior
10 15 20 25 30 35 40
0
50
100
150
IgG injection (mL)
IgA(mg/dl)
pH5.7
pH6.0
pH6.3
Feed
Content of IgA in
flow-through
fractions
Wash
IgA binding on gel
Limited pH impact
Gel robustness
Limited impact of pH on IgA
content in flow-through at
least until a volume of
injection of ca. 20 mL of Ig

Phase 1a Result
Impact of pH on IgM chromatographic behavior
10 15 20 25 30 35 40
0
5
10
15
20
25
IgG injection (mL)
IgM(mg/dl)
pH5.7
pH6.0
pH6.3
Feed
Content of IgM in
flow-through fractions
Wash

 Even under worst-case conditions (crude IgG feed and high
linear flow rate of 185 cm/hr):
✓ Obvious preferential and reproducible binding (and
therefore removal) of IgA at pH 5.7-6.3 allowing to
purify IgG in a negative chromatographic mode
✓ Removal of IgM is also apparent, but apparently less
efficient than for IgA, possibly due to IgM content in this
CA-IgG fraction
 No apparent impact of pH on IgG recovery (flow-through)
and IgA and IgM removal (binding), clearly proving
Fractogel® EMD TMAE (M) resin robustness in the pH 5.7 to
pH 6.3 range
 The data allowed to select loading conditions for further
studies (phase 1b)
Phase 1a
Conclusions
19 Chromatographic Strategies for IVIG Purification - Part 2 | 29.11.18

Optimal Loading Volume Determination
Considering the content in IgA and IgM of the CA-IgG fraction
IgG recovery reaches
90% at IgG injection
volume of 12.5 mL
IgA removal reaches
80% with a volume of
IgG of 12.5 mL
30 20 12.5 10
0
20
40
60
80
100
IgG Injection volume (mL)
IgGRecovery(percentage)
IgG recovery
30 mL 89%
20 mL 93%
12.5 mL 94%
10 mL 90%
30 20 12.5 10
0
20
40
60
80
100
IgARemoval(percentage)
IgA removal
30 mL 46%
20 mL 59%
12.5 mL 84%
10 mL 89%
IgM removal reaches
90% at IgG injection
volume of 12.5 mL
30 20 12.5 10
0
20
40
60
80
100
IgMRemoval(percentage)
IgM removal
30 mL 39%
20 mL 60%
12.5 mL 90%
10 mL 92%

Optimal loading volume
12.5 mL CA-IgG chosen
corresponding to about 137 mg of total proteins
Volume used for phase 1b
Phase 1a Conclusions

Phase 1b
Analysis of IgG purity
using optimized
conditions: 10 cycles
pH 6.0
12.5 mL
185 cm/hr

Phase 1b Results
Consistent chromatographic patterns over 10 cycles
Cycle 1
2017Nov23no001:10_UV 2017Nov23no001:10_Cond 2017Nov23no001:10_pH
2017Nov23no001:10_Fractions
0
500
1000
1500
2000
mAu
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 ml
1 2 3 4 5 6 7 Waste 8 9 Waste
Cycle 10
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
500.0 510.0 520.0 530.0 540.0 550.0 ml
32 33 34 35 Waste 36 38 Waste

Phase 1b Results
The concentration of total protein (Biuret)
Sample Total Protein (mg/mL)
Feed 11
C1 Flow-through 4
C2 Flow-through 4
C3 Flow-through 4
C4 Flow-through 4
C5 Flow-through 4
C6 Flow-through 4
C7 Flow-through 4
C8 Flow-through 4
C9 Flow-through 4
C10 Flow-through 4
Sample Total Protein (mg/mL)
C1 Wash 6
C2 Wash 7
C3 Wash 7
C4 Wash 7
C5 Wash 6
C6 Wash 7
C7 Wash 7
C8 Wash 7
C9 Wash 7
C10 Wash 6

Phase 1b Results
Recovery of IgG, IgA, IgM
C
y
c
le
1
C
y
c
le
2
C
y
c
le
3
C
y
c
le
4
C
y
c
le
5
C
y
c
le
6
C
y
c
le
7
C
y
c
le
8
C
y
c
le
9
C
y
c
le
1
0
0
2 0
4 0
6 0
8 0
1 0 0
Mean IgG recovery in the flow-
through is 94.18% (89.66-97.66%)
C
y
c
le
1
C
y
c
le
2
C
y
c
le
3
C
y
c
le
4
C
y
c
le
5
C
y
c
le
6
C
y
c
le
7
C
y
c
le
8
C
y
c
le
9
C
y
c
le
1
0
0
2 0
4 0
6 0
8 0
1 0 0
Mean IgA content in the wash is
84.00% (82.18-86.18%)
C
y
c
le
1C
y
c
le
2C
y
c
le
3C
y
c
le
4C
y
c
le
5C
y
c
le
6C
y
c
le
7C
y
c
le
8C
y
c
le
9
C
y
c
le
1
0
0
2 0
4 0
6 0
8 0
1 0 0
Mean IgM content in the wash is
89.82% (81.81-99.80%)
*IgG (ELISA) *IgA (ELISA) *IgM (ELISA)

Phase 1b Results
IgG, IgA and IgM in feed, flow-through and wash:
10 cycles with good reproducibility
Blue: IgG
Red: IgA
Green: IgM
• Good reproducibility of Fractogel® EMD TMAE (M) resin to remove IgA and IgM from CA-IgG fraction
• Improved IgG purity

Phase 1b Results
IgG subclass distribution in feed: Flow-through remains throughout
10 cycles
• Reproducible content in all 4 subclasses
• Slight decrease in subclass 4 content in the flow-through (subclass 4 has a pI closer to that of
IgA, so more likely to be adsorbed on the resin)
Mean of 10 cycles
58.40% IgG1
38.58% IgG2
1.37% IgG3
1.63% IgG4

Phase 1b Results
SDS PAGE shows reproducible removal of contaminants in the wash
Flow-through
←IgG
←LC
←HC
Flow-through
Non-reduced Reduced

Phase 1b Results
FXIa content
Sample Factor XIa Concentration (mg/dL)
Feed
<0.0000575
Cycle 1 Flow-through
* FXIa activity tested by FXIa Hyphen
• Chromatographic
separation on
Fractogel® EMD
TMAE (M) resin did
not induce FXIa
activity
• The process does
not induce
detectable risks of
thrombogenicity
linked to activation
of Factor XI

Phase 1b Results
Thrombin generation assay (TGA)
*Thrombin generation assay (Technoclone)
RC High
Thrombin
[nmol/L]
Time to Peak
[min.]
Velocity
Index
AUC
Feed 16.16 35.00 0.52 94.11
Feed (diluted 2.46X) ND ND ND ND
Cycle 1 Flow-through ND ND ND ND
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
0 10 20 30 40 50 60 70 80 90 100
nMThrombin
time [min]
Thrombin
• Slight tendency for thrombin generation in
the feed
• No thrombin generation was reproducibly
found in the 10 flow-through cycles
• Fractogel® EMD TMAE (M) chromatography
resin may contribute to the removal of
factors (proteolytic enzymes) responsible
for thrombin generation
• Spiking experiments with pro-thrombotic
factors may be considered to confirm

 Even under worst-case conditions (crude IgG feed + high linear flow
rate):
✓ Powerful removal of IgA and IgM at 12.5 mL CA-IgG/mL of gel
(=75-90 mg IgG)
 Robustness of Fractogel® EMD TMAE resin at pH 6.0:
✓ Consistent removal of IgA and IgM (constant proportion present
in the flow-through)
 The wash fraction is proportionally enriched in IgA and IgM,
confirming adsorption on TMAE
 Good consistent recovery of IgG subclasses (only limited decrease in
the proportion of IgG subclass 4)
 Improvement in IgG purity and removal of IgA and IgM observed by:
✓ Immunonephelometry
✓ ELISA
✓ Zone electrophoresis
✓ SDS-PAGE under reduced and non-reduced conditions, which also
reveals removal of various protein contaminants
 No generation of thrombogenicity (based on FXIa and TGA data)
during Fractogel® EMD TMAE chromatography resin, and potential
removal of thrombogenic factors
Phase 1b
Conclusion
using CA-IgG

• Control fraction II IgG containing more purified IgG,
199 mg of IgG/mL of gel loading
• 5 cycles done
• Checked:
• Chromatographic profile
• IgG recovery: 97.50% (range 91.76-103.72%)
• IgA removal: 92.16% (range 88.64-94.18%)
• IgM removal: 66.75% (range 59.77-74.14%)
• IgG/A/M distributions in Feed/Flow-through/Wash
over 5 cycles show good consistency
• IgG subclasses distribution consistent, with IgG4
dropped a little
Phase 1b Conclusion
Control experiment using Fraction II IgG
Flow-through Cycle 5
Flow-through Cycle 5

Cycle 1
0
200
400
600
800
1000
mAu
0 20 40 60 80 ml
1 2 3 4 5 6 7 Waste 8 1011 Waste
Flow-through
Wash
Regeneration/CIP
Chromatographic profile obtained with purified IgG
Phase 1b Conclusion
Evidence of adsorption of contaminants on the resin

Phase 2
Tests of stability and
robustness of resin
performance up to 200
cycles pH 6.0
12.5 mL
185 cm/hr

Phase 2
Test conditions and a typical chromatographic profile
Buffer Contents pH
Conductivity
(mS)
Pre-
equilibration
500 mM sodium acetate 6 20.8
Equilibration 25 mM sodium acetate 6 1.97
Wash 500 mM sodium acetate 4.5 22.8
Regeneration
1.5 M NaCl in 250 mM
sodium acetate
4.5 >30.00
CIP
0.5 M NaOH (odd run
numbers)
1 M NaOH (even run
numbers)
Sample pH Conductivity (mS)
Cycle 1-IgG 6 1.737
Cycle 201-IgG 6 1.744
2018Feb11no004:10_UV 2018Feb11no004:10_Cond
2018Feb11no004:10_pH 2018Feb11no004:10_Fractions
2018Feb11no004:10_Logbook
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
0 20 40 60 80 ml
Break point 2 Break point 5 Break point 9 Break point 10 Break point 12
1 3 4 5 6 Waste 10 Waste
Flow-through
Wash Regeneration/CIP
Cycle 1

Phase 2
Consistent chromatographic profile throughout 201 cycles
Cycle 11
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
0 20 40 60 80 ml
Cycle 31
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
0 20 40 60 80 ml
Cycle 512018Feb23no002:10_UV 2018Feb23no002:10_Cond
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
0 20 40 60 80 ml
Cycle 71
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
0 20 40 60 80 ml
Cycle 91
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
0 20 40 60 80 ml
1 3 5 6 Waste 10 Waste
Cycle 111
2018Mar02no002:10_UV 2018Mar02no002:10_Cond
2018Mar02no002:10_pH 2018Mar02no002:10_Fractions
2018Mar02no002:10_Logbook
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
0 20 40 60 80 ml
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
0 20 40 60 80 ml
Cycle 131 Cycle 151
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
0 20 40 60 80 ml
Cycle 181
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
0 20 40 60 80 ml
Cycle 201
0
500
1000
1500
2000
mAu
4.0
6.0
8.0
10.0
0.0 20.0 40.0 60.0 80.0 ml
Break point 2 Break point 5 Break point 9 Break point 10 Flow 0.0 ml/min
12 3 4 5 6 7 8 10 Waste

Phase 2
Total protein consistent throughout 201 cycles
Sample Total Protein (mg/mL) Albumin (mg/mL)
C1 Feed 12 < 2
C201 Feed 10 < 2
C1 Flow-through 4 < 2
C201 Flow-through 4 < 2

mean IgG recovery in the flow-
through is 94.22% (range 91.09-
97.78%)
Phase 2
IgG Recovery, IgA Removal, IgM Removal consistency test
C
1C
1
1
C
2
1
C
3
1C
4
1
C
5
1
C
6
1
C
7
1C
8
1
C
9
1C
1
0
1C
1
1
1C
1
2
1C
1
3
1C
1
4
1C
1
5
1C
1
6
1C
1
7
1C
1
8
1C
1
9
1C
2
0
1
0
5 0
1 0 0
1 5 0
C
1C
1
1
C
2
1
C
3
1C
4
1
C
5
1
C
6
1
C
7
1C
8
1
C
9
1C
1
0
1C
1
1
1C
1
2
1C
1
3
1C
1
4
1C
1
5
1C
1
6
1C
1
7
1C
1
8
1C
1
9
1C
2
0
1
0
5 0
1 0 0
1 5 0
mean IgA content in the wash is
96.16% (range 87.48-107.15%)
C
1C
1
1
C
2
1
C
3
1C
4
1
C
5
1
C
6
1
C
7
1C
8
1
C
9
1C
1
0
1C
1
1
1C
1
2
1C
1
3
1C
1
4
1C
1
5
1C
1
6
1C
1
7
1C
1
8
1C
1
9
1C
2
0
1
0
5 0
1 0 0
1 5 0
mean IgM content in the wash is
96.89% (range 87.22-108.06%)

Phase 2 Results
Reproducible removal of IgA and IgM over the 201 cycles performed

Phase 2 Results
Pie chart of IgG, IgA and IgM in Wash for 201 cycles
C 1 W a s h C 1 1 W a s h C 2 1 W a s h C 3 1 W a s h C 4 1 W a s h C 5 1 W a s h
C 6 1 W a s h C 7 1 W a s h C 8 1 W a s h C 9 1 W a s h C 1 0 1 W a s h C 1 1 1 W a s h
C 1 2 1 W a s h C 1 3 1 W a s h C 1 4 1 W a s h C 1 5 1 W a s h C 1 6 1 W a s h C 1 7 1 W a s h
C 1 8 1 W a s h C 1 9 1 W a s h C 2 0 1 W a s h

Phase 2 Results
Reproducible IgG subclass distribution in the purified flow-through
over the 201 cycles performed
C101 Flow-through
Feed

Percentage(%)
C
1
F
e
e
d
C
2
0
1
F
e
e
d
C
1
F
T
(c
o
n
c
.
3
X
)C
2
0
1
F
T
(c
o
n
c
.
3
X
)
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
G a m m a + B e ta
A lp h a 1
A lp h a 2
A lb u m in
Zone electrophoresis
C1
Feed
C201
Feed
C1 FT
(conc. 3X)
C201 FT
(conc. 3X)
Albumin 1.7% 1.8% 1.1% 1.4%
Alpha 2 9.2% 9.4% 4.3% 3.2%
Alpha 1 4.4% 2.8% 0.3% 0.3%
Gamma +
Beta
84.7% 86.0% 94.3% 95.1%
C1 Feed C201 Feed
C1 Flow-through
(conc. 3X)
C201 Flow-through
(conc. 3X)

Phase 2
SDS-PAGE
Non-Reduced Reduced

• Chromatographic separation on Fractogel® EMD
TMAE (M) resin did not induce the generation of
FXIa activity
• Process does not increase possible risks of
thrombogenicity linked to activation of Factor XI
• These findings are constant over 201 cycles
Phase 2
Factor XIa assay and TGA activity
Sample Factor XIa concentration (mg/dL)
Cycle 1 Feed
<0.0000575
Cycle 201 Feed
0,00
0,50
1,00
1,50
2,00
0 10 20 30 40 50 60 70 80 90 100
nMThrombin
time [min]
Cycle 1 Feed (2.25X)
Cycle 201 Feed (2.25X)

Phase 2
Conclusion
1
Fractogel® EMD TMAE (M) resin demonstrates to have
excellent resistance for over 201 cycles in its capacity
to robustly remove IgA and IgM from a crude CA-IgG
preparation, under good recovery of IgG and without
affecting the IgG subclass distribution
Excellent resistance to cleaning/sanitization cycles
2
The chromatographic purification process does not
generate detectable thrombin activity and may remove
thrombogenic factors present in the CA-IgG feed
3
Optimal loading conditions would have to be
determined by each plasma fractionator based on the
level of IgA or IgM present in the IgG fraction to be
purified

How to
systematically
set up an IVIG
purification
strategy
How to integrate
chromatography
In a downstream
process of IVIG
How to evaluate the
quality of purified
IVIG
How to confirm
the robustness of
an upgraded IVIG
purification
process

Questions?
Dr. Thierry Burnouf
thburnouf@gmail.com
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. The vibrant M, Cogent and Fractogel are trademarks of Merck KGaA,
Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via
publicly accessible resources.

Chromatography: Chromatographic strategies for IVIG purification – Part 2

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Chromatography: Chromatographic strategies for IVIG purification – Part 2