Participate in the interactive webinar now: http://bit.ly/ViralClearanceWebinar Is viral clearance a hurdle to implementing continuous processing? We’ll share virus spiking alternatives that may pave the way for effectively evaluating viral clearance by chromatography steps in a continuous process. Explore our webinar library: www.merckmillipore.com/webinars

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Exploring Viral
Clearance for
Continuous
Processes
Kathryn Martin Remington, Ph.D.
05 November 2019

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

3. Agenda
The evolution of continuous processing and viral clearance
strategies
Viral clearance for batch vs. continuous processing
Five methods
1
2
3
Impact of spiking methods
Potential future studies
4
5

4. The Evolution of
Bioprocessing
Current
2019-2020+

5. 5
Continuous Processing Offers Advantages
Reduced
Footprint
Reduced
COGs
Increased
Product
Quality
Flexibility

6. 6
Potential Hurdles to Continuous Processing
On-Line Analytics
Viral Clearance

7. 7
Viral Safety Strategy of Continuous Processes
Safe sourcing and testing
of raw materials
Verify absence
of viral contaminants
at appropriate stages
Verify capacity of manufacturing
process to remove or inactivate
potential viral contaminants

8. 8
Viral Clearance for Continuous Processes
Verify absence
of viral contaminants
at appropriate stages
Verify capacity of manufacturing
process to remove or inactivate
potential viral contaminants
• What is the appropriate scale
down model?
• Is a multi-column chromatography
system needed for clearance
studies?
• Will my VC CRO have access to a
multi-column chromatography
system?
• Will I need to demonstrate that
my VC CRO’s system is a valid
representation of my downscaled
system?

9. 9
Viral Clearance for a Batch Process
9
Spiked Column Load
Input Virus
Leftover Virus
Cleared Virus
• Develop scale down model
of individual step
• Verify that scale down
model represents full scale
process step
• Spike step load intermediate
with each virus, one virus at
a time
• Perform small scale process
step
• Assay spiked load and
product-containing fraction
for infectious virus

10. 10
Output from Batch vs. Continuous Process
10
5
10
15
0 2 4 6 8 10 12
Output from well
mixed tank
Protein A CEX AEX Chromatograms from Godawat
et al. 2015. J Biotechnol 213:13

11. 11
What is the Best scale down
model to use to
demonstrate clearance
across a continuous
process?

12. 12
Lab Scale Model of Continuous Process
 Downscaled model of continuous process using inline spiking and sampling
 Experimental design and data analysis are more complex
 May be difficult to implement in GLP VC CRO; specialized equipment and experienced operators
required
 Is this continuous and constant level of virus introduction represent the way virus
might be introduced to the column?

13. 13
Batch study of each step using worst case conditions identified for
continuous process
• Design, implement and analyze using existing equipment and methods
• Use a risk-based approach to conduct experimental design
• Need a thorough scientific understanding of the process step and those parameters that
might impact viral clearance

14. 14
Anion Exchange (AEX) CHromatography
 For mAb process, negatively charged impurities are
removed by electrostatic interactions
 Load at pH 7-8, below the pI of the mAb
 99% of mAb (+) flows through; DNA (-), HCP (-), viruses
(-) are retained
 pH and conductivity have been widely studied and shown
to impact virus binding
 Can be a robust viral clearance step
Spike Introduction
 Under the conditions of pH and conductivity that typically
achieve 4-5 log10 virus reduction, different methods of
spike introduction were evaluated
 It is unclear whether potentially contaminating virus would
be introduced as a bolus or in a homogeneous stream.
Various options were evaluated
+
+
+
+
+
+
-
+
++
+
-
-
-

15. 15
Experimental Procedure
• 80 mL of mAb feed loaded
onto column
• Targeted 8 log10 total MMV
per column run; although
measured concentrations within
0.2 log10 of target
• Flowthough collected in
four (4) fractions; rinse
collected as a fifth fraction
• Each fraction assayed for
infectious virus
• A percentage of each
fraction pooled and pool
assayed for infectious
virus
• Standard ÄKTA® Pure system
• Eshumuno ® Q AEX resin
• In-House mAb 2
• Column load at pH 8.5, 6.0
mS/cm

16. Method 1 – “Batch” Spiking
16

17. Method 1 – “Batch” Spiking
17
8 log10MMV
80 mL mAb
System
Pumps
Inlet Valves
Sample
Inlet Valve
Sample
Pump
Mixer
Column
Valve
Column
Fractions 1-5
Conductivity
Monitor
UV
Monitor
Outlet

18. Method 1 – “Batch” Spiking
18
5.5 log10 LRV
Initial virus load

19. Method 2 – Virus Pulse
19

20. Method 2 – Virus Pulse
20
System
Pumps
Inlet Valves
Sample
Inlet Valve
Sample
Pump
Column
Valve
Column
Fractions 1-5
UV
Monitor
Conductivity
Monitor
Outlet
40 mL mAb
40 mL mAb
10 mL MMV
in process
intermediate
Method
mAb Pool A mAb Pool B
mAb Conc
(g/L)
Volume
(CV)
Virus
Concentration
(log10 TCID50/mL
mAb Conc
(g/L)
Volume
(CV)
Virus
Concentration
(log10 TCID50/mL
2 8.3 ± 0.2 18 8.3 ± 0.2 2 6.9 ± 0.2

21. Method 2 – Virus Pulse
21
Method LRV
1 - batch 5.5
2 – virus pulse ≥ 6.1
Initial virus load
Method 1 Method 2

22. Method 3 – Virus Peak
22

23. Method 3 – Virus Peak
23
80 mL mAb
System
Pumps
Inlet Valves
Sample
Inlet Valve
Sample
Pump
Column
Valve
Column
Fractions 1-5
UV
Monitor
Conductivity
Monitor
Outlet
1mL MMV in
process
intermediate
introduced midway
through load
Method
mAb Pool A
mAb Conc
(g/L)
Volume
(CV)
Virus Concentration
(log10 TCID50/mL
3 9.2 ± 0.1 20 7.9 ± 0.1

24. Method 3 – Virus Peak
24
Initial virus load
Method 1 Method 2 Method 3
Method LRV
1 - batch 5.5
2 – virus pulse ≥ 6.1
3 – virus peak ≥ 6.2

25. Method 4 – Virus and mAb Peak
25

26. Method 4 – Virus and mAb Peak
26
72 mL mAb
System
Pumps
Inlet Valves
Sample
Inlet Valve
Sample
Pump
Mixer
Column
Valve
Column
Fractions 1-5
Conductivity
Monitor
UV
Monitor
Outlet
1mL MMV
8 mL mAb
introduced
midway
through load
Method
mAb Pool A mAb Pool B
mAb Conc
(g/L)
Volume
(CV)
Virus
Concentration
(log10 TCID50/mL)
mAb Conc
(g/L)
Volume
(CV)
Virus
Concentration
(log10 TCID50/mL)
4 3.1 ± 0.1 18 56 ± 2 2 7.2 ± 0.3

27. Method 4 – Virus and mAb Peak
27
Initial virus load
Method 1 Method 2 Method 3 Method 4
Method LRV
1 - batch 5.5
2 – virus pulse ≥ 6.1
3 – virus peak ≥ 6.2
4 – virus/mAb peak 5.5

28. Method 5 – mAb Peak
28

29. Method 5 – Constant Virus with mAb Peak
29
72 mL mAb
System
Pumps
Inlet Valves
Sample
Inlet Valve
Sample
Pump
Mixer
Column
Valve
Column
Fractions 1-5
Conductivity
Monitor
UV
Monitor
Outlet
4mL MMV
8 mL mAb
introduced midway
through load Method
mAb Pool A mAb Pool B
mAb Conc
(g/L)
Volume
(CV)
Virus
Concentration
(log10 TCID50/mL
mAb Conc
(g/L)
Volume
(CV)
Virus
Concentration
(log10 TCID50/mL
5 3.0 ± 0.2 18 55 ± 1 2

30. Method 5 – Constant Virus with mAb Peak
30
Initial virus load
Method 1 Method 2 Method 3 Method 4 Method 5

31. Log Reduction Values
Method
Log10 Viral
Reduction
1 Batch 5.5
2 Virus Pulse ≥ 6.1
3 Virus Peak ≥ 6.2
4
Virus/mAb
Peak
5.5
5 mAb Peak 4.9

32. Host Cell Protein Reduction
Method
% HCP
Removal
1 Batch 97
2 Virus Pulse 96
3 Virus Peak 96
4
Virus/mAb
Peak
95
5 mAb Peak 96

33. Column Feed in Continuous Process
 Output from a column in a continuous process is typically in pulses,
so that rather than a homogeneous pool, the load for the next
column may contain concentration fluctuations
Variation in Virus Concentration
 Homogeneous mixture of virus and column feed (Method 1) resulted
in removal of 5.5 log10 virus
 Virus introduced as a concentrated pulse was completely removed by
the AEX column (≥6.1 log10 reduction)
 Virus introduced in a more concentrated, sharp peak was also
completely removed by the AEX column (≥6.2 log10 reduction)
Variation in mAb Concentration
 A simulated elution peak of mAb containing a highly concentrated
peak of virus, did not impact virus reduction; 5.5 log10 virus was
removed
 Removal of virus introduced by inline spiking at a constant
level was not impacted by a pulse of concentrated mAb;
4.9 log10 virus reduction was achieved
33
Impact of Spiking Method on Virus Reduction by AEX

34.  This approach can be used to evaluate other potentially fluctuating
conditions: pH, salt concentration, multiple peaks of mAb or virus
 Steps with less robust clearance can be evaluated to determine
whether the manner in which the spike is introduced or
fluctuations in mAb concentration have an impact on viral
clearance
 These methods can also be used to evaluate other types of
chromatography such as bind/elute or flow-through membrane
chromatography
34
Potential Future Studies

35.  Regulators do expect a risk-based approach and solid scientific
justification which evaluates all of the potential conditions that might be
observed in a continuous process.
 Developing scale down models of continuous processes that can be used
in a CRO is challenging & requires specialized equipment and trained
personnel.
 The potential exists for evaluating steps of a continuous process using
standard chromatography systems and more traditional study
designs
 Our studies have advanced the field of continuous bioprocessing providing
better understanding of the viral clearance implications.
35
Viral Clearance for Continuous Processes
Let us partner with you to evaluate steps in your
continuous process!

36. Technical Consultant
Field Technology Management
kathy.remington@milliporesigma.com
240 447 6280
Kathryn Martin Remington, Ph.D.
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