Biotherapeutic manufacturing processes are at greater risk of contamination than classic small molecule processes and therefore require different control strategies. Understanding the source, options for control, and potential impact of bioburden throughout downstream biopharmaceutical processes is beneficial to process developers, production operators and pharmaceutical microbiologists. Process designs that reduce the risks of bioburden contamination will decrease process related failures and the resulting painful, time-consuming investigations. Register for our webinar here: https://bit.ly/2SyJrgz

1. Merck KGaA
Darmstadt, Germany
April 30, 2020
Somasundaram Gopalakrishnan
Senior Technical Consultant Asia Pacific
Technology Management, Process Solutions
Strategies to Address
Bioburden Control in
Downstream Processing

2. Strategies to Address Bioburden Control in Downstream Processing
The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.

3. Objectives
1 Understand the impact of
bioburden excursions
2 Recognize sources of
bioburden
3 Develop strategies to
mitigate risk
Strategies to Address Bioburden Control in Downstream Processing

4. 1
Impact of
uncontrolled
bioburden
Strategies to Address Bioburden Control in Downstream Processing

5. 30
Percent
1- 6Months
1-14Million Euro
Percent of process deviations
caused by contamination*
Length of time to complete
an investigation
Operations cost
*Sources Langer 2013, Wiebe 2014
Biologics in-process contamination
Strategies to Address Bioburden Control in Downstream Processing

6. 30
Percent
1- 6Months
1-14Million Euro
*Sources Langer 2013, Wiebe 2014
Biologics in-process contamination
Impact
 Productivity losses
 Material replacement costs
 Batch loss
 Interruption of product supply
 Delay in clinical development
Strategies to Address Bioburden Control in Downstream Processing

7. 2 Sources of
bioburden
Strategies to Address Bioburden Control in Downstream Processing

8. Microbial
Byproducts
Endotoxin
Exotoxin
Lipopeptides
Flagellin
DNA
Extracellular proteases
Bioburden
Bacteria
Fungi
Mycoplasma
Virus
TSE Agents
What?
Downstream bioburden and byproducts
Scope
Strategies to Address Bioburden Control in Downstream Processing

9. Facility & Environment
Equipment
Processes
Materials
Utilities
Personnel
Each source
contributes
to the
process
bioburden
profile
Sources of bioburden
Origin?
Staphylococcus
Bacillus
Non-fermenting
Gram Negative rods
Aspergillus
Source: Public domain CDC/
Robert Simmons
Strategies
to Address
Bioburden
Control in
Downstream
Processing

10. Downstream
Where?
Risk profile and control strategies differ throughout the process
Secondary
Clarification
Chromatography
Protein A
Final FillingFinal Sterile
Filtration
Concentration
& Formulation
Bulk Storage
and Transport
Viral
Inactivation
Chromatography
CEX
Virus Filtration
Clearance
Ultrafiltration /
Diafiltration
Bioreactor
Primary
Clarification
MCB WCB Seed Train
Raw Materials
Filtration Bioburden
Reduction
Bioburden
Reduction
Chromatography
AEX
Bioburden
Reduction
Bioburden
Reduction
Final Fill
Risk Areas
Upstream
Downstream
Strategies to Address Bioburden Control in Downstream Processing

11. Case Study
Bioburden excursions in the Protein A Pool
Chrom
Protein A
Bulk
Storage and
Transport
Viral
Inactivation
Chrom
CEX
Virus
Filtration
Clearance
Ultrafiltration
Diafiltration
Bioburden
Reduction
Bioburden
Reduction
Chrom
AEX
Bioburden
Reduction
Bioburden
Reduction
Situation
• Spore-forming bioburden alert-level excursions in the
Protein A pool over several campaigns
Root Cause
• Failure to recognize a trend in the pattern of excursions
• Sanitization solution was not sporicidal
• Sub-optimal sanitization process
Corrective and Preventative Actions
• Scale down studies with a new sanitizer and
optimization of sanitization conditions
• Process scale verification
Strategies to Address Bioburden Control in Downstream Processing

12. Case Study
Bioburden action-level excursions in the UF/DF step
Chrom
Protein A
Bulk
Storage and
Transport
Viral
Inactivation
Chrom
CEX
Virus
Filtration
Clearance
Ultrafiltration
Diafiltration
Bioburden
Reduction
Bioburden
Reduction
Chrom
AEX
Bioburden
Reduction
Bioburden
Reduction
Situation
• Bioburden and endotoxin exceeded action levels in
multiple batches
• Intensive investigation of the process and support areas
Root Cause
• Bioburden formation in the TFF cassettes due to inadequate
cleaning and storage processes
Corrective and Preventative Actions
• Improve cleaning and storage processes
• Sterilization or sanitization of buffer tanks
• Assessment of the water for injection (WFI) system and
transfer lines
• Introduction of bioburden reducing filters
• Validation of hold times and storage conditions
• Revision of bioburden limits based on process capability
Buffer, Sanitizer, and
Storage Solutions
Operations
WFI
Operation
Suvarna K. et. al. “Case Studies of Microbial Contamination in Biologic Product”,
American Pharmaceutical Review 14(1) January/February 2011.
Strategies to Address Bioburden Control in Downstream Processing

13. Case Study
Sporadic bioburden action-level excursions
Chrom
Protein A
Bulk
Storage and
Transport
Viral
Inactivation
Chrom
CEX
Virus
Filtration
Clearance
Ultrafiltration
Diafiltration
Bioburden
Reduction
Bioburden
Reduction
Chrom
AEX
Bioburden
Reduction
Bioburden
Reduction
Situation
• Sporadic mixed bioburden excursions at multiple
points in the downstream process
Root Cause
• Aseptic connections of equipment and sampling devices
Corrective and Preventative Actions
• Short term:
• Retrained operators in aseptic techniques
• Long term:
• Reduced the number of aseptic connections
• Implemented sterile to sterile connectors and steam
to sterile connectors.
• Introduced a facility-wide sterile sampling system
Strategies to Address Bioburden Control in Downstream Processing

14. Case Study
Bulk solution contamination with Bacillus species
Chrom
Protein A
Bulk
Storage and
Transport
Viral
Inactivation
Chrom
CEX
Virus
Filtration
Clearance
Ultrafiltration
Diafiltration
Bioburden
Reduction
Bioburden
Reduction
Chrom
AEX
Bioburden
Reduction
Bioburden
Reduction
Situation
• Prefiltration bioburden load was 20 x the
specification
• Bacillus species suggested a steam-in-place issue
Root Cause
• Equipment design: improper pipe slope resulted in
cold spots that were insufficiently sterilized
Corrective and Preventative Actions
• Discard (scrap) the batch
• Redesign piping
• Requalify SIP (steam-in-place) cycle
• New processes will use single-use storage systems
Strategies to Address Bioburden Control in Downstream Processing

15. Many routes for microbial ingress
Downstream bioburden excursions are often the result of
 Improper cleaning, storage, or sanitization
 Suboptimal system design
 Aseptic connections
 Sampling
 Lapses in aseptic technique
Intensive risk assessments could have prevented many of these
contaminations or excursions
Key Points
Sources of bioburden
Strategies
to Address
Bioburden
Control in
Downstream
Processing

16. 3 Mitigation
Strategies
Strategies to Address Bioburden Control in Downstream Processing

17. Assess
Mitigate
Monitor
Risk Mitigation Strategies
Strategies to Address Bioburden Control in Downstream Processing

18. The risk profile varies from upstream to final fill
Risk Hammock
Upstream Downstream Finish and Fill
Operations
Risk
High
Low
Aseptic AsepticBioburden Controlled
“The level of effort, formality and
documentation of the quality risk
management process should align
with the level of risk”
(ICH Q9A)
Strategies
to Address
Bioburden
Control in
Downstream
Processing

19. Assess
Mitigate
Monitor
Assess Risk
Assess extent of risk, ability to detect,
and frequency of occurrence
Strategies to Address Bioburden Control in Downstream Processing

20. Microbiological Profile
Get to know the bioburden in your neighborhood
Who are
we?
How many
of us? Will we cause
you
trouble?
Where are
we
from?
Strategies to Address Bioburden Control in Downstream Processing

21. Focus Objective
Fault
Tree
Analysis
FMEA HACCP
Statistical
Methods
Ishekawa
(Fish
Bone)
Risk
Assessment
Risk Identification   
Risk Analysis  
Risk Evaluation X  
Risk
Control
Risk Reduction X   X
Risk Acceptance X  X 
Risk Review X 
Risk Communication X X  
Characterize Process Risk
Multiple tools are available
Adapted from Roenninger S., Hertlein M. “Which Risk assessment fits best?”
Logfile No 15 November 2011 1-4 Maas & Peither AG GMP Publishing
 Very suitable
Limited suitability
X Not suitable
Strategies
to Address
Bioburden
Control in
Downstream
Processing

22. What parts of the process could
introduce bioburden?
(Hu)Man Mother Nature Material
MethodMachine
Hygiene
Training
Air Handling Water
Sanitizers
Disinfectants
Buffers
Chromatography
Gaskets
Water System
Storage Buffer
Load cells
Mixer
Mixing
Compounding
Hold Time
Room Cleaning
Transfer
Transfer
Testing
Sampling
Weighing
Measurement
Calibration
Microscopy
Humidity
Tubing / Piping
Filters
Resin
Tanks
Pump
Transfer Panel
Steam Generator
Aseptic Technique
Clean room
Filtration
Pump
Air compressor
Pressure
Flow Rate
Time
Temperature
Enumeration
Insects
Animals i.e. Rodents
Microorganisms
Mass
Columns
Experience
ChromatogramData Acquisition
Packing Installing
Bioburden profile
Strategies to Address Bioburden Control in Downstream Processing

23. CMC Biotech Working Group: A-Mab: A case Study in Bioprocess Development
Score
• Probability of occurrence
• Severity
• Ability to detect
• Criticality
Prioritize
How can I put the risks into
perspective?
Strategies to Address Bioburden Control in Downstream Processing

24. Key Points
 Characterize the microbial profile of the process
 Utilize a combination of assessment tools
 A cross-functional team is crucial to the process
 Your bioburden risk mitigation strategy should address
 Patient safety
 Drug supply
 Business risk
Assess Risks
Strategies to Address Bioburden Control in Downstream Processing

25. Assess
Mitigate
Monitor
Mitigation Considerations
Eliminate source or reduce
likelihood of occurrence
Strategies to Address Bioburden Control in Downstream Processing

26. Bioburden profile
 What microorganisms are present?
 How many?
 Variation over time?
 Toxin producing?
 Spore formers?
Material Origin
 Material consistency
 Supplier transparency
 Quality management system
 Quality philosophy
Material Characteristics
 Growth Promoting
 Bacteriostatic
 Bactericidal
Risk Mitigation
Material Considerations
Mitigate
Strategies to Address Bioburden Control in Downstream Processing

27. Risk
Cell disruption results in byproducts
Sanitizer Modes of Action
Bacterial spores
• Spore coat penetration
Vegetative bacteria
• Cell wall and cytoplasmic disruption
Fungi
• Cell wall and cytoplasmic disruption
Virus
• Capsid and nucleic acid damage
Risk Mitigation Sanitization and Storage
Modes of action of sanitizers
Reduce bioburden load
Material selection
Containment
• Single-use
• Closed sampling
• Pre-packed columns
Removal
• Filtration
Mitigate
Strategies to Address Bioburden Control in Downstream Processing

28. Prevent Contamination by Containment
Implement Single-use Systems and Closed Sampling
Eliminate
bioburden
contribution
Prevent
microbial
ingress
Minimize
process
validation
Mitigate
Strategies to Address Bioburden Control in Downstream Processing

29. Upstream Process
 Bioreactor protection
 Drug supply continuity
 Business risk mitigation
Downstream Process
 Bioburden Reduction
 Assure drug substance purity
Final Fill
 Regulatory requirements
 Drug product sterility assurance
 Assure patient safety
What is your objective?
Risk-based filter selection What filtration option is best
for my process?
Strategies to Address Bioburden Control in Downstream Processing

30. Goal: Minimize in-process bioburden
• No requirement to validate sterility
• Reduce bioburden proliferation
• With other controls, bioburden reduction
filters may provide sufficient process safety
• Prefiltration can increase filter capacity and
improve efficiency
Bioburden reduction filters (0.45 or 0.2 m)
• May be sufficient for intermediate processing steps
• May be used as prefilters upstream of sterilizing-
grade filters
• Manufacturers may validate bacterial reduction, but
not sterilizing performance
Sterilizing-grade rated filters (0.2 m)
 Manufacturers must validate bacterial removal
 Traditionally used for intermediate processing steps
Downstream Filtration
Minimize Bioburden What filtration option is best for
my downstream process?
Strategies to Address Bioburden Control in Downstream Processing

31. Mitigate Risks
Key Points
 A downstream bioburden control risk mitigation strategy addresses
drug supply continuity, business risk, and patient safety
 The act of sanitization, while effective, can release microbial
byproducts into the process
 Single-use systems prevent microbial ingress and reduce bioburden
contribution
 Closed sterile sampling prevents false-positive tests
 Filtration choice is dependent upon risk assessment, capacity, and
cost per liter
Mitigate
Strategies to Address Bioburden Control in Downstream Processing

32. Assess
Mitigate
Monitor
Mitigation Strategy: Monitor
Monitor trends
Strategies to Address Bioburden Control in Downstream Processing

33. Downstream Monitoring
What do I test for? Where? Why?
Chromatography
Protein A
Bulk Storage
and Transport
Viral
Inactivation
Chromatography
CEX
Virus Filtration
Clearance
Ultrafiltration /
Diafiltration
Bioburden
Reduction
Bioburden
Reduction
Chromatography
AEX
Bioburden
Reduction
Bioburden
Reduction
Bioburden
Virus
Mycoplasma
Endotoxin
V
M
B
E
B
E
B
E
B
E
V
E
V
E
B
E
B
E
M
Monitor
Strategies to Address Bioburden Control in Downstream Processing

34. Monitor
How much is too much?
“Unlike non-sterile dosage forms, there are no recommended bioburden levels provided in
regulatory guidelines or compendia for the [downstream] protein purification processes of
biologic or other biopharmaceutical products, therefore, manufacturers are responsible for
setting bioburden control levels for biologic production processes.”
“The BPOG Bioburden Working Group conducted a member survey of bioburden action levels
and found that the majority of biologic processes action levels were set between 1-10
CFU/mL.”
Bain, D. “Microbial Monitoring For Biological Drug Substance Manufacturing: An Industry Perspective”
BioPhorum Operations Group. 2015.
Strategies to Address Bioburden Control in Downstream Processing

35.  Downstream processing is considered a low-bioburden controlled
process
 Bioburden monitoring is a supporting tool in a risk mitigation
strategy
 Appropriately set action and alert levels coupled with data trending
permit timely responses
Key Points
Mitigation Strategy
Monitor
Strategies to Address Bioburden Control in Downstream Processing

36. Summary
Strategies to Address Bioburden Control in Downstream Processing

37. Strategies to address bioburden control in downstream processing
Final Points
Bioburden excursions present real and under-appreciated risks
For every publically disclosed contamination event, there are countless others
Implications of bioburden excursions can be significant
Investigational and decontamination costs, production downtime, lost revenues due to drug
stock-out, regulatory fines, loss of consumer confidence
Multiple approaches are needed to provide the required control
Low bioburden processes require as much attention as sterile processes.
A robust risk mitigation strategy uses a three-pronged approach of
 Risk assessment
 Mitigation
 Monitoring
Strategies to Address Bioburden Control in Downstream Processing

38. Langer, E. “Biopharm Shows Signs of Maturity”. Pharmaceutical Manufacturing, in: Biopharmaceutical manufacturing Excellence Within a
Rapidly Changing Landscape, pp 20-25, 2013. Link
Wiebe, M. “Update on the CAACB Virus Contamination Project”. presented at 2014 PDA/FDA Virus & TSE Safety Conference 2014.
von Wintzingerode, V. “Biologics Production: Impact of Bioburden Contaminations of Non-Sterile Process Intermediates on Patient Safety and
Product Quality”. American Pharmaceutical Review. 20(3). April 2017. Link
Suvarna K. et. al. “Case Studies of Microbial Contamination in Biologic Product”, American Pharmaceutical Review 14(1) January/February
2011. Link
K. Suvarna “Case Studies of Microbial Contamination in Biologic Product” presented at PDA 5th Annual Global Conference on Pharmaceutical
Microbiology, October 2010.
Roenninger S., Hertlein M. “Which Risk assessment fits best?” Logfile No 15 November 2011 1-4 Maas & Peither AG GMP Publishing.
Oliver J. “3D risk assessment model”, Journal of Validation Technology, Autumn 2008, page 70-76. Link
Bain, D. “Microbial Monitoring For Biological Drug Substance Manufacturing: An Industry Perspective” BioPhorum Operations Group. 2015.
Link
CMC Biotech Working Group: A-Mab: A case Study in Bioprocess Development. Link
PDA, “Technical Report No. 69 Bioburden and Biofilm Management in Pharmaceutical Manufacturing Operations. Parenteral Drug Association.
2015.
Strategies to address bioburden control in downstream processing
Recommended Reading
Strategies
to Address
Bioburden
Control in
Downstream
Processing

39. Acknowledgments
• Kerry Roche Lentine, Director, Technology Management, Global Growth Programs
Strategies to Address Bioburden Control in Downstream Processing

40. Somasundaram G.
© 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. The vibrant M is 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.
Senior Technical Consultant
somasundaram.g@emdgroup.com