This presentation reviews the results of a study in which the authors investigated the effects of poly-diallydimethylammonium chloride (pDADMAC) flocculation and clarification on the performance and longevity of protein A resin. To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: http://www.merckmillipore.com/mlab

1. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.
Philip Szymanski, Akshat Gupta, and Dana Kinzlmaier
August 6th, 2020
Improvement in protein A resin lifetime by
implementing poly-diallyldimethylammonium
chloride (pDADMAC) flocculation coupled
with gradient depth filtration

2. Agenda
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
2
1 Background
2 Results
3 Wash Optimization and Scale-up/Implementation
4 Summary

3. 3 Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
Background
Relevance
2
1
mAb producing cell lines are becoming more productive
 Flocculants are one tool to combat the bottleneck created by higher cell densities
 Insufficient investigation into the effects of flocculant-based clarification on subsequent
downstream unit operations
Protein A Resin Performance
 Large contributor to the total cost of purification raw materials
 Purification process costs are sensitive to resin longevity
Kelley, “Very Large Scale Monoclonal Antibody Purification: The Case for Conventional Unit Operations”. Biotechnol. Prog. 2007, 23, 995-1008

4. Background
pDADMAC
Poly(diallyldimethylammonium chloride)
 Polymeric, cationic flocculant (binds negatively charged species)
 Promotes the formation of flocs in the 30-40µm range
 Couples with Clarisolve® 40MS depth filters for effective clarification
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
4

5. Background
Feed Streams
 mAb02 cell culture dosed with
pDADMAC at 30pg/cell
 Titer = 1.64 g/L
 Hand-packed Omnifit® column
 HETP = 0.054cm
 102mm bed height
 3.49mL CV
 Naïve DBC10% = 41.3 mg/mL CV
Eshmuno® A Capture
Chromatography
Clarisolve® 40MS Clarification
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
5
 mAb02 cell culture
 Titer = 1.69 g/L
 Hand-packed Omnifit® column
 HETP = 0.043cm
 101mm bed height
 3.46mL CV
 Naïve DBC10% = 43.9 mg/mL CV
Eshmuno® A Capture
Chromatography
Millistak+® D0HC → X0HC
Clarification
Flocculated Feed
Non-Flocculated Feed

6. Background
Feed Stream Impurities
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
6
0.0E+00
2.0E+05
4.0E+05
6.0E+05
8.0E+05
1.0E+06
1.2E+06
1.4E+06
[HCP]
ppm
Concentration of Host Cell Proteins in Clarified Cell Culture
Non-flocculated Flocculated
Flocculated Feed contains 31% less HCPs

7. 7 Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
Background
Chromatography Method
Step Buffer/Solution CV RT (min)
Equilibration 25mM Tris, 150mM NaCl, pH 7.2 5 3
Load HCCF with 0.08% NaN3 ~20 4
Wash 25mM Tris, 500mM NaCl, pH 7.2 5 3
Elution 100mM Acetate, pH 3.0 5 3
Strip 150mM H3PO4 5 3
Strip Wash-out 25mM Tris, 150mM NaCl, pH 7.2 1 3
Regeneration
(skip if sanitization)
0.1M NaOH 5 3
Sanitization* 0.4M NaOH 5 3
Sanitization Wash-out 25mM Tris, 150mM NaCl, pH 7.2 5 3
• Loaded to 80% of DBC10%
• Sanitization every 10th cycle
• Elution peak collection from
100mAU to 96mAU at 280nm
(2mm flow cell)
*every 10th cycle

8. Background
Resin Life Endpoints
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
8
DBC10% falls below 80% of naïve column
Pressure increases >100%
• Arrived at this number using pressure v. flow data for Eshmuno®
A resin
• 20cm packed bed height yields 1.5 bar differential pressure at
400 cm/hr linear velocity
• Assume 4 bar limit for process scale chromatography system,
25% safety factor
• Resulting loss of product
Or

9. • Titer Measurements
• HPLC method using POROS™ analytical protein A column
• Yield Measurements
• Absorbance at 280nm using SoloVPE UV-VIS spectrophotometer
• Pressure Drop measured offline using PendoTECH pressure transducers coupled with our data
acquisition system for greater resolution
• Host Cell Protein Concentrations
• Plate-based 3G CHO HCP ELISA kit from Cygnus, Catalog F550
• Leached Protein A Concentrations
• Plate-based ELISA from Repligen, Catalog 9000-1
Background
Analytical Assays
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
9

10. Agenda
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
10
1 Background
2 Results
3 Wash Optimization and Scale-up/Implementation
4 Summary

11. 11 Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
Results
Evolution of Eluate Volume and ΔP
0.5
0.7
0.9
1.1
1.3
1.5
1.7
1.9
0 50 100 150 200
mAb
Eluate
Pool
Volume
(CV)
Cycle no.
Trend of mAb Eluate Pool Volume
Non-flocculated Flocculated
-5
0
5
10
15
20
25
30
35
40
45
0 50 100 150 200
Column
Backpressure
Increase
(%)
Relative
to
New
Column
Cycle no.
Trend of Column Backpressure
Non-flocculated Flocculated
Maximum pressure increase:
<10% for flocculated feed
>40% for non-flocculated feed

12. 12 Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
Results
Non-flocculated Feed Chromatogram - Elution Peak
Chromatogram overlay comparing UV and pH traces of:
No significant changes in peak shape or width, no artifact peaks
Load Wash Elution
Elution
Blue curves: 1st cycle using 0.1M NaOH regeneration step
Brown curves: 100th cycle using 0.4M NaOH sanitization step

13. 13 Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
Results
Flocculated Feed Chromatogram - Elution Peak
Chromatogram overlay comparing UV and pH traces of:
No significant changes in peak shape or width, no artifact peaks
Load Wash Elution
Elution
Blue curves: 1st cycle using 0.1M NaOH regeneration step
Brown curves: 160th cycle using 0.4M NaOH sanitization step

14. 14 Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
Results
Evolution of Yield and Dynamic Binding Capacity
0%
20%
40%
60%
80%
100%
0 50 100 150 200
mAb
yield
Cycle no.
mAb Yield Trend
Non-flocculated Flocculated
95%
75%
80%
85%
90%
95%
100%
105%
0 50 100 150 200
Relative
DBC
10%
Cycle no.
Relative DBC10% Trend
Non-flocculated Flocculated
33% increase in number of cycles based
on DBC10% Trends

15. Results
Eluate Quality - Host Cell Protein Clearance
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
15
Average [HCP]
Non-
flocculated
Feed
3,735 ppm
Flocculated
Feed
734 ppm
0
0.5
1
1.5
2
2.5
3
3.5
1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Log
clearance
Cycle no.
Host Cell Protein Clearance
Non-flocculated Flocculated
Average = 2.5
Average = 3.1

16. Results
Eluate Quality - Leached Protein A
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
16
Average [Leached
Protein A]
Non-
flocculated
Feed
6.1 ppm
Flocculated
Feed
8.3 ppm
0
5
10
15
20
25
30
1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Concentration
(ppm)
Cycle no.
Leached Protein A levels
Non-flocculated Flocculated

17. • Residual pDADMAC concentration in the protein A eluate samples was determined using LC-MS
• Several samples were analyzed without pre-treatment, but a few underwent a protein precipitation pre-
treatment using methanol to decrease signal noise and interference
• Column: Phenomenex® Aeris Widepore XB-C18, 4.6mm x 150mm x 3.6um
• Samples were spiked with pure pDADMAC to determine limit of detection (LOD) and limit of
quantitation (LOQ)
• LOD and LOQ were calculated for each sample based on signal to noise ratio (S/N)
Results
Quantitation of Residual pDADMAC - Method
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
17
Example of a total ion chromatogram

18. • LOQ ranged from 1.46 – 15.33 ppm
Results
Quantitation of Residual pDADMAC – Preliminary Results
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
18
Sample Pre-treatment Sample Name LOD (ppm) LOQ (ppm) Result
Protein Precipitation
Null Control Load <LOD
Null Control Cycle 1 <LOD
pDADMAC Load <LOD
pDADMAC Cycle 1 Eluate <LOD
pDADMAC Cycle 10 Eluate <LOD
pDADMAC Cycle 20 Eluate <LOD
None
pDADMAC Cycle 30 Eluate 1.31 4.38 <LOQ
pDADMAC Cycle 40 Eluate 0.66 2.19 <LOD
pDADMAC Cycle 50 Eluate 0.92 3.07 <LOQ
pDADMAC Cycle 60 Eluate 0.92 3.07 <LOQ
pDADMAC Cycle 70 Eluate 1.15 3.83 <LOQ
pDADMAC Cycle 80 Eluate 0.54 1.8 <LOD
pDADMAC Cycle 90 Eluate 0.44 1.46 <LOQ
pDADMAC Cycle 100 Eluate 1.31 4.38 <LOD
pDADMAC Cycle 110 Eluate 0.71 2.36 <LOD
pDADMAC Cycle 120 Eluate 0.71 2.36 <LOD
pDADMAC Cycle 130 Eluate 0.84 2.78 <LOD
pDADMAC Cycle 140 Eluate 0.48 1.61 <LOD
pDADMAC Cycle 150 Eluate 3.07 10.22 <LOD
pDADMAC Cycle 160 Eluate 3.07 10.22 <LOD
Buffer Control 4.6 15.33 <LOD
Residual levels in all samples below LOQ

19. Agenda
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
19
1 Background
2 Results
3 Wash Optimization and Scale-up/Implementation
4 Summary

20. Wash Optimization and Scale-up/Implementation
Wash Optimization for HCP removal
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
20
Control (25mM Tris,
150mM NaCl, pH 7.2)
Trace Buffer Yield [HCP] ppm
Yellow
25mM Tris, 500mM
NaCl, pH 7.2
99.3% 1009
Green 50mM Citrate, pH 5.0 96.8% 949
Purple
50mM Acetate, pH
5.0
92.8% 746
Yield vs. Purity
Holstein, “Protein A Intermediate Wash Strategies”. BioProcess International. Feb 2015, 13(2), 52-58

21. Wash Optimization and Scale-up/Implementation
Scale-up performance
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
21
Column
Column
Volume (mL)
Loading (g
mAb/L resin)
Eluate
Volume (CV)
Yield [HCP] ppm
0.66cm ID 3.39 30.0 1.4 96% 4,091
3.2cm ID 116.6 28.2 1.4 97% 4,889
• Used control feed due
to limitations
• Slightly different
chromatography
methods (differing
block lengths)
Brown trace =
0.66cm ID
Blue Trace =
3.2cm ID
Comparable
performance

22. Wash Optimization and Scale-up/Implementation
COGs Benefits compared to standard filtration train
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
22
Millistak+® HC Filters Clarisolve® Filters
Surface installed 26 m² 7 m²
Clarification train
Two-step clarification:
D0HC → X0HC
One-step clarification:
pDADMAC → 40 MS
Consumables cost
Equivalent (including Flexware™ assemblies and
flocculant)
Water for flush 2600 L 700 L
€
V=1800 L
CHO
mAb 1.2-1.3 g/L
17.84 x 106 TC/mL
56.2% viability
30% safety factor
Increase in resin
longevity
translates to
>20% decrease
in protein A resin
consumables cost
Additionally

23. Summary
Key Takeaways
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
23
1
2
3 Reduced impurity levels post protein A chromatography
compared to traditional clarification
The use of pDADMAC and Clarisolve® 40MS for clarification
extended column’s DBC10% lifespan by 33% (40 cycles)
Significantly more stable backpressure profile over 160 cycles
compared to traditional clarification
Boost in Protein A Resin Performance

24. Acknowledgements • Santosh Rahane
• Kristina Cunningham
• Derek Silva
• Kara Pizzelli
• Renato Azevedo
• Kellie Grogan
• Sebastian Hickert
• Tim Mueller
Improvement in protein A resin lifetime by implementing pDADMAC flocculation | P Szymanski
24

25. Plan your visit today.
MerckMillipore.com/mlab

26. The vibrant M, Clarisolve, Eshmuno, Flexware, Millstak+, and M Lab 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.
© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.