A Turn-Key Flow-Through-Mode Purification Process to improve Quality and Safety of Plasma IgG

The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
A Turn-key flow-through-mode
purification process to improve
quality and safety of plasma IgG
Collaboration work with Taipei Medical University
Prof. Thierry Burnouf, Vice Dean, Director of the International PhD
Program in Biomedical Engineering,
College of Biomedical Engineering,
Taipei Medical University
Josephine Cheng
Senior Consultant, Core modalities APAC,
Bioprocessing strategy
Webinar Oct. 12, 2021

The life science business
of Merck KGaA, Darmstadt,
Germany operates as
MilliporeSigma in the U.S.
and Canada

Agenda
1
2
Introduction
Study results
3 Discussion & Conclusions

Population
growth
Immunoglobulin G is the leading plasma-derived medicine
54%
16%
11%
9%
9%
IgG
Factors
Albumin
Protease Inhibitors
Others
2020 Global
Plasma Market
(28 B USD)
58%
15%
9%
10%
8%
IgG
Albumin
Factors
Protease Inhibitors
Others
2025 Global
Plasma Market
(39 B USD)
7.9%
5.4%
3.1%
7.6%
4.3%
Source: Plasma fractionation markets report 2020, marketsandmarkets.
ON/Off
label use
SCIG
Adoption
Webinar Turnkey flowthrough IGG purification | Oct. 2021
◼ Example IgG Usages:
- Primary
immunodeficiency
- Secondary
immunodeficiency
- Autoimmune and
inflammatory diseases
- Hyperimmune diseases
- Convalescent IgG (COVID)
◼ Listed as Essential
medicines by WHO
4

Shortage
Improvement
on Quality &
Productivity
A generic, easy-to-operate,
flowthrough-mode purification
process that provides scalable &
robust purification with enhanced
productivity and quality IGG fitting
for therapeutic usage.
High market demands drives exploration in process optimization
Accessibility
to LMIC Quality criteria:
• Virus safety
• Low IgA & IgM contamination
• Low FXI/XIa
• Lack of Hemolytic effect
• Lack of chemicals used for virus
inactivation
5

Poll Question #1

From Plasma donation to patient adminstration
Experimental flow with key steps used in purification
A generic, easy-to-operate,
flowthrough-mode purification
process that provides scalable
& robust purification with
enhanced productivity and
quality IGG fitting for
therapeutic usage.
Source: JC et. al. Process steps for fractionation of IgG depleted of IgA,
isoagglutinins, and devoid of in vitro thrombogenicity. Blood Transfusion 2021,
DOI 10.2450/2021.0159-21
7

Materials and Methods used in IgG purification process
8
No. Process step Description
1 Caprylic Acid Treatment/centrifugation
Added 5% caprylic acid to precipitate non-IgG protein and centrifuged to remove
precipitates to generate starting materials representing worse case of Fraction
I+II+III in plasma fractionation process.
2 Batch TFF UF/DF
Concentrated and diafiltrated against the chromatographic equilibration buffer
using Pellicon® 3 Biomax (30 kDa, A screen) via tangential flow filtration
(TFF) method prior to the subsequent chromatography purification steps.
3 Anion exchange (AEX) chromatography
Fractogel® EMD TMAE (M) anion exchange chromatography resin for primarily
purification. Major IgA and IgM was removed.
4
Pre-affinity chromatography SPTFF
concentration
Concentration was performed using Pellicon® 3 Biomax (30 kDa, A screen)
via Single-Pass TFF technology, to achieve an optimal loading concentration of
>40 mg/mL for following affinity chromatography purification.
5 Affinity chromatography
Eshmuno® P anti-A and anti-B, two distinct, affinity-based chromatography
resins, were used to remove blood type anti-A and anti-B isoagglutinin.
6
S/D treatment and S/D removal by C18
reverse phase chromatography
Solvent/detergent (S/D) treatment was applied to the IgG batches for enveloped
virus inactivation. The LiChroprep® RP-18 (40-63 µm) column was used in a
flow-through mode to remove the S/D.
7 SPTFF final concentration
Single-Pass TFF technology using Pellicon® 3 Biomax (30 kDa, D screen) to
achieve final target concentration of 200 mg/mL.

Analytical Steps involved along the process
DOI 10.2450/2021.0159-21
9

Optimization of anion-
exchange Chromatography
step with Fractogel®
TMAE(M) resin

Evaluating optimal chromatographic parameters for IgA and IgM
removal
Fractogel™ TMAE resin - Anion Exchange chromatography
Evaluation of pH effect on Fractogel® TMAE(M) resin binding capacity.
Typical chromatograph for a
flow-through mode anion
exchange chroamtography.
25mM Sodium Acetate buffer,
with 185cm/h flow rate.
Learn More with our webinar:
Chromatography: Chromatographic
strategies for IVIG purification - Part 2
• No detectable impact IgA/IgM binding
and IgG purification from 3 pH values.
• Optimal pH for loading identified @ pH
6.0
12

IgG, IgA and IgM in feed, flow-through and wash:
10 cycles (data shown previously) & 200 cycles with good
reproducibility
1. Purity IgG
increases
from 87% to
99% at small
and pilot scale.
2. Recovery avg.
96.89±7.06%
3. 200 cycles
test with
standard
cleaning
conditions
confirms the
robustness
Lab-scale anion exchange chromatography step: robustness test
13

IGG Subclasses distribution in 200 cycle tested to be
similar
lab-scale
Subclasses IgG 1 IgG 2 IgG3 IgG4
Feed 61.43% 34.49% 1.46% 2.62%
Flowthrough 61.83±3.35% 35.35±3.32% 1.42±0.09% 1.39±0.08%
• Satisfactory preservation of
the IgG subclass distribution.
14

Summary AEX:
1. Optimal pH for loading identified @ pH 6.0
2. Purity increase for IgG from 82% to 99, in
pilot scale almost 100% purity was reached.
3. 200 cycles test with standard cleaning
conditions confirms the robustness.
4. No major changes in IgG subclasses.
5. No in vitro thrombin generation detected.
Pilot scale 10 L batches study
confirms Fractogel™ as major step
removing IgA and IgM
15

Anti-A and anti-B
Isoaglutinin removal

Blood type isoaglutinins increases risk for hemolysis and
provides some limitation on plasma collection
1. High dose treatment for
Immunomodulation patients.
2. Additional safety measure for
fractionators to avoid failed batches/
decrease isoaglutinins content.
3. Safe product even for high-dose
administration.
17

Eshmuno® P Anti-A (FT) Eshmuno ® P Anti-B (FT)
Robust reduction of the blood-type specific isoagglutinins
Affinity chromatography
8 to 16 times reduction in Anti-A titer 16 to 32 times reduction in Anti-B titer
*samples tested at 30mg/ml concentration from 1st SPTFF (6X) step to the last step.
Feed loaded onto column with
25mM Sodium Acetate buffer
in flowthrough mode, 3 min
residence time.
18

S/D virus inactivation
and removal by
reverse-phase
chromatography

Classical Solvent detergent proven to effectively reduced model
viruses with single-use bags in time as short as 5 mintues
Human IgG: 0.3% TnBP + 1% TX100 LRV Results
Virus Device
LRV at Incubation Time (min)
5 30 60 360
XMuLV
Mobius® 1 ≥ 5.5 ≥ 5.3 ≥ 5.3 ≥ 5.4
Mobius® 2 ≥ 5.5 ≥ 5.3 ≥ 5.3 ≥ 5.5
BVDV
Mobius® 1
Mobius® 2
≥ 4.5
≥ 4.4
≥ 4.4
≥ 4.6
≥ 4.6
≥ 4.4
≥ 4.5
≥ 4.5
Publication: Hsieh YT, Mullin L, Greenhalgh P, Cunningham
M, Goodrich E, et al. Single-use technology for
solvent/detergent virus inactivation of industrial plasma
products. TRANSFUSION 2016;56;1384–1393.
0.3% TnBP + 1% Triton X-100 provides > 4-5
LRV in time as short as 5 minutes
Learn More with our webinar: Solvent Detergent Viral Inactivation using
S.U Technology in Blood Fractionation Processes
21

Cell viability assay of 3T3 cells provides quick understanding
for S/D residual level and safety of the purified IgG
Cell toxicity data of the SD-treated IgG following C18 chromatography
Cells were treated with 0-130 ppm S/D-spiked IgG for establishment of a standard curve (A) and flow
through of S/D treated-IgG after C-18 column (B). Cells were incubated for 24 hours with the S/D-spiked
IgG or the C18 flow-through of the SD-treated IgG. The cell viability was analyzed by CCK-8 assay. FT, flow-
through; ppm, part per million; S/D, solvent/detergent.
NIH-3T3 cells treated with 2
ppm S/D-spiked IgG
(standard curve) started to
evidence a 4.3% decrease in
viability. The flow-through of
C18 (FT18) corresponding to
a loading with 18 mL per ml
of C18 packing evidenced
signs of cytotoxicity
indicating that 17 mL of S/D
spiked IgG was the
maximum volume of SD-
treated IgG to loaded onto 1
mL C18.
17ml of S/D spiked IgG/
ml packed C18 resin
When S/D concentration
~ 4ppm, viability of
cells starts to decrease
DOI 10.2450/2021.0159-21
22

Efficient removal of S/D by Licroprep® C18 (40 – 63um)
sorbent to non-detectable level
Virus Inactivation and removal by C18 RP-chromatography
A. Typical chromatography for FT mode S/D-IGG running through C18 column.
B. TnBP residual tested on GC-MS with results < 1ppm, Triton X-100 residual tested by HPLC with results < 2ppm,
showing a robust removal with the loading quantity defined (6ml S/D-IGG/ml C18 packed resin), with capacity
tested at 17 ml S/D-IGG/ml C18 resin)
Residual Triton X-100 of SD-IgG
(Ratio of resin and loaded IgG)
Batch 3
(1mL:6mL)
C18 <2 ppm
SPTFF-5X <2 ppm
Residual TnBP of SD-IgG
(Ratio of resin and loaded IgG)
Batch 3
(1mL:6mL)
C18 <1 ppm
SPTFF-5X <1 ppm
A. B.
23

Filters used for Clarification, Bioburden reduction, and
sterile filtration
Filter Name Filter Area Membrane Pore Size Composition & Symmetry Type
Millipore Express® SHC Optiscale®
25 mm
3.5 cm2 0.5 / 0.2 μm PES*, asymmetric Membrane filter
Durapore® 0.22 μm
Optiscale® 25 mm
3.5 cm2 0.22 μm PVDF**, symmetric Membrane filter
Milligard® PES Optiscale® 25 mm 3.5 cm2 1.2 / 0.2 μm PES, asymmetric Membrane filter
Millistak+® HC μPod A1HC 23 cm2
<0.5 μm
(Nominal pore size)
Cellulose fibers with inorganic filter aid
(DE65 + ED70)
Mixed esters of cellulose (RW01)
Depth filter
25

Trial Schematic of filters tested after each major unit operation
26
Steps Pre-filter Sterile Filter
Post Caprylic Acid Treatment) Milligard® PES 1.2/0.2 μm
- Durapore® 0.22 μm
- Millipore Express® SHC 0.5/0.2 μm
Post UF/DF
Milligard® PES 1.2/0.2 μm
Millistak+® A1HC
Post CA-IgG passed through Fractogel
resin
NA
Post CA-IgG 6X SPTFF concentration Milligard® PES 1.2/0.2 μm
Post CA-IgG passing through Anti-B
resin
NA
Post S/D- pure IgG passing through
C18 resin
Post Pure IgG 5X SPTFF NA
Vmax™ methods
(constant Pressure)
Pmax™ methods
(Constant flow)

27
Step.1 : Post-Caprylic Acid
Treatment/centrifugation Step.2 : Post-Batch TFF UF/DF Step.3 : Post-AEX chromatography Step.4 : Post-SPTFF concentration
Step.5 : Post-Affinity chromatography
Step.6 : Post-C18 reverse phase
chromatography
Step.7 : Post-SPTFF final concentration

Bioburden filters &
clarification filters used
in the study:
• Millstak+® A1HC
• Milligard® PES
1.2/0.2
• Millipore Express®
SHC 0.5/0.2
• Durapore® filter0.22
Various intermediate filtration steps reducing bioburden
Prefilters provide significant protection effect to sterile
filters and can be used stand alone
0
500
1000
1500
0 10 20 30 40 50
Throughput
(L/m
2
)
Time (min)
Milligard® PES 1.2/0.2 μm -> Express® SHC 0.5/0.2 μm
Milligard® PES 1.2/0.2 μm -> Durapore® 0.22 μm
Millipore Express® SHC 0.5/0.2 μm
Durapore® 0.22 μm
0
200
400
600
800
1000
1200
0 4 8 12 16 20
Throughput
(L/m
2
)
Time (min)
Millistak+® HC A1HC -> Express® SHC
0.5/0.2 μm
Observations:
• w/o prefilter- Millipore
Express ® SHC generally
performs better
• w/ prefilter – sometimes
Durapore® performs better
• Prefilter shows significant
protecting effect / reducing
membrane area needed for
sterile filters.
• Millistak® A1HC clarification
filter reduced significantly the
particles/ precipitation.
Steps Selected Prefilters
(Recovery %)
Selected Sterile
Filter
Recovery (%)
Post CA MPES (100%) Durapore® filter > 99
Post UF/DF Millistak+® A1HC Millipore Express® SHC ~ 100
Post AEX Millipore Express® SHC ~ 100
Post 1st SPTFF MPES (100%) Durapore® filter > 96
Post Anti A/B Millipore Express® SHC ~ 100
Post C18 resin MPES (100%) Millipore Express® SHC ~ 98
Post final SPTFF Millipore Express® SHC ~ 86
28

Ultrafiltration &
Single-pass TFF
optimization

Ultrafiltation/diafiltration successfully removed caprylic
acid from the IgG fraction, data showed consistent
removal from 3 pilot scale batches
• 6x diafiltration was carried out under
constant volume mode at ~2x VCF.
• pH and conductivity from permeate were
taken at each diafiltration volume (DN). As
shown in the diafiltration profiles, target
pH and conductivity were reached at 5-
6 DN. Typically 2 DN will be added as
safety factor.
• Initial concentration : 8.74 mg/mL
• Final concentration: 16.16 mg/mL
30

31
Filter
Pellicon® 3, Biomax®
30KD, A screen
Area 0.33
Initial conc. 11.76 mg/ml Initial vol. ~4 L
Final conc. 74.62 mg/ml Final vol. ~670 mL
Feed flux 0.57 LMM TMP 11-12psi
Conversion(%) ~83%
SPTFF successful concentrated the sample to 74 mg/ml, achieved optimal loading concentration
for subsequent anion exchange step. Also reduce time and volume for chromatography step.
Retentate pressure excursion Feed Flux excursion Permeate Flux TMP excursion
Single-Pass TFF optimization provides options for inline concentration

A gentle method for high concentration ultrafiltration
Single-Pass Tangential Flow Filtration
Summary:
1. Sequential optimization to identify
parameters to reach target concentration
20%. (200 mg/ml)
2. SPTFF before Eshmuno® P Anti-A/B step
reduced loading time, buffer consumed,
also improved the AC performance.
3. SPTFF for final concentration successfully
provided a gentle (low shear force),
scalable, high recovery, and easy-to-
operate method for high concentration
ultrafiltration.
Process Parameters
Trial volume 726 ml Feed flux 0.01 LMM
Target VCF 5 x TMP 11-12 psi
Conversion 80 %
Retentate
Pressure
~10
Final
volume
145.2 ml
LMM: feed flow rate in L per min per total square meters of membrane area;
TMP: transmembrane pressure.
32

Satisfying concentration effect and IgA/IgG removal ability
Overall Quality test results
57
14 21 14
72 67 62
43
200
0
50
100
150
200
250
Total protein
concentration (mg/ml)
10.92 8.47 16.16 11.76
74.62 65.91 61.37
42
198
0
50
100
150
200
250
IgG concentration
(mg/ml)
1.88
1.45
2.69
0.0034 0.022 0.0182 0.0179 0.0114 0.0619
0
0.5
1
1.5
2
2.5
3
IgA concentration
(mg/ml) 0.7
0.42
0.52
0.0002 0.0003 0.0003 0.0003 0.0001 0.0006
0
0.2
0.4
0.6
0.8
IgM Concentration
(mg/ml)
34

TGA & Zone Electrophoresis confirming purity increases along the
process
Overall Quality test results
Thrombin generation assay (TGA) conducted on IgG fractions over the various stages of purification,
indicating Fractogel TMAE removing pro-thrombogenic factors.
Lag Phase (min) Thrombin (nM) Time to Peak (min) Velocity Index AUC
CPP 4.75 ±0.50 52.16 ± 14.73 27.25 ±19.92 6.98 ± 6.93 954.20 ± 325.20
CA-IgG 4.00 ±0.00 27.20 ±5.92 44.50 ±0.58 0.67 ± 0.15 291.24 ± 155.68
Fractogel® BDL BDL BDL BDL BDL
SPTFF BDL BDL BDL BDL BDL
*BDL= below detection limits
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Recovery from major steps (%)
35

Comparable purity to market product
Quality check benchmarking market product
Fractions Non-IgG IgG
Unit % %
5XSPTFF-B1 4.8 95.2
5XSPTFF-B2 1.1 98.9
5XSPTFF-B3 0.6 99.4
Privigen® IgG 0.3 99.7
Zone electrophoresis confirmed the high
purity of the final IgG. The purity of batch
3 (5X SPTFF) reaches almost 100%.
Albumin
IgG
Albumin
IgG-HC
IgG-LC
M: Protein
marker
1:CPP
2: CA-IgG
3: Fractogel ® FT
4: Anti-A-FT
5: Anti-B-FT
6: C18
7:SPTFF-5X
8:Privigen IgG
SDS-PAGE under non-reducing (left) and
reducing (right) conditions showing the
step wise purification process, purity was
enhanced especially after AEX step.
*5 µg of protein loaded on to 4-12% Bis-Tris SDS-PAGE
36

Conclusions
1. Milligard® PES 1.2/0.2 prefilters effectively
reduced the filtration area needed for sterile
filters e.g. Millipore Express® SHC or Durapore®
filters
2. Clarification using Millistak+® HC A1HC to
facilitate downstream purification.
3. Fractogel® TMAE(M) anion exchange
chromatography for efficient removal of IgA and
IgM, with well maintained IgG subclasses.
Proof of concept for a generic
process with intensified
processing:
4. Eshmuno® P anti-A and Eshmuno® P anti-B
chromatography for robust removal of anti-A
and anti-B agglutinins.
5. S/D used in virus inactivation can be efficiently
removed using Licroprep® C18 Reverse-phase
chromatography.
6. Use of SPTFF as a mild and robust approach to
concentrate IgG to a target of 20%
concentration.
7. Recovery from 92 – 100% for each step
resulting in an overall process recovery of
greater than 70% under a worst-case scenario,
with opportunities to improve further with
additional optimization.
Such flow-through process can be monitored reliably by controlling
key process parameters, ready to be scalable, and can be applied
for various IgG products such including polyvalent IgG,
hyperimmune, or convalescent immunoglobulins.
38

Publications
ISBT GBS Working Party Workshop presentation:
https://www.isbtweb.org/working-parties/global-blood-
safety/workshop-recordings
39
Blood Transfusion
publicaion:
http://www.bloodtra
nsfusion.it/articolosi
ng.aspx?id=001177

Poll Question #3

Acknowledgements
Taipei Medical University, Taiwan (TMU):
• Prof. Thierry Burnouf
• Yu-Wen Wu
• Chen-Yun Wang
• Cheum Lam Hong
Merck:
• Sharon ShangJung Wu
• Karen Waiyu Chan
• Leo Xun Liao
• Xisheng Cao
• Bin Wang

Acknowledgement to team
working behind the scene
Lynn Neild, Ravin Gami, Jessica Torres, Patryk Kelley
(MilliporeSigma, USA), Jennifer Kercher, Nina Weis,
Andreas Stein, Andre Kiesewetter, Michael Schulte
(Merck KGaA, Germany), Manuel Brantner (Merck
Chemicals and Life Science GesmbH, Austria), Anissa
Boumlic, Carole Inglevert (Millipore SAS, France),
Sandra Hon (Merck Pte Ltd, Singapore) for their support.
We thank the Taipei Blood Center for plasma supply
(Guandu, Taiwan Blood Service Foundation, Taiwan).

Thank You !
Q&A
Merck, Millipore, Eshmuno, Fractogel, Durapore, Milligard, LiChroprep, Millipore Express,
Millistak and the vibrant M 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.
© October 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

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