Strategies to Address

Bioburden Control in
Downstream Processing
Somasundaram Gopalakrishnan
Senior Technical Consultant Asia Pacific
Technology Management, Process Solutions

April 30, 2020

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

Strategies to Address Bioburden Control in Downstream Processing

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

 Impact of
1 uncontrolled
bioburden

Strategies to Address Bioburden Control in Downstream Processing

Biologics in-process contamination

30
Percent of process deviations
caused by contamination*

Length of time to complete

Percent an investigation

Operations cost

1- 6 Months
1-14
Million Euro
*Sources Langer 2013, Wiebe 2014

Strategies to Address Bioburden Control in Downstream Processing

Biologics in-process contamination

Impact

30
 Productivity losses
 Material replacement costs
 Batch loss
Percent
 Interruption of product supply
 Delay in clinical development

1- 6 Months
1-14
Million Euro
*Sources Langer 2013, Wiebe 2014

Strategies to Address Bioburden Control in Downstream Processing

 2 Sources of
bioburden

Strategies to Address Bioburden Control in Downstream Processing

What?
Downstream bioburden and byproducts

Bioburden Microbial
Byproducts

Bacteria Endotoxin
Scope
Fungi Exotoxin
Mycoplasma Lipopeptides
Virus Flagellin
TSE Agents DNA
Extracellular proteases

Strategies to Address Bioburden Control in Downstream Processing

Origin?
Sources of bioburden
Aspergillus
Source: Public domain CDC/
Robert Simmons

Facility & Environment

Equipment
Bacillus
Each source
Processes contributes
to the
Materials process
bioburden
Staphylococcus

Utilities
profile

Personnel Non-fermenting
Gram Negative rods

Strategies
to Address
Bioburden
Control in
Downstream
Processing
 Where?
Risk Areas
Risk profile and control strategies differ throughout the process

Upstream Downstream

MCB WCB Seed Train

Raw Materials

Primary Secondary Viral Bioburden Chromatography

Filtration Bioreactor Bioburden Chromatography
Clarification Clarification Inactivation Reduction CEX
Reduction Protein A

Downstream
Final Fill

Bioburden Chromatography Virus Filtration Ultrafiltration / Bioburden Bulk Storage Concentration Final Sterile Final Filling
Reduction AEX Clearance Diafiltration Reduction and Transport & Formulation Filtration
Strategies to Address Bioburden Control in Downstream Processing
 Case Study
Bioburden excursions in the Protein A Pool

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
Bioburden Chrom Viral Bioburden Chrom
Reduction Protein A Inactivation Reduction CEX • 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
Bioburden Chrom Virus Ultrafiltration Bioburden Bulk
Reduction AEX Filtration Diafiltration Reduction Storage and
Clearance Transport

Strategies to Address Bioburden Control in Downstream Processing

 Case Study
Bioburden action-level excursions in the UF/DF step

Situation
• Bioburden and endotoxin exceeded action levels in
multiple batches
• Intensive investigation of the process and support areas
WFI Buffer, Sanitizer, and
Operation Storage Solutions
Operations Root Cause
• Bioburden formation in the TFF cassettes due to inadequate
cleaning and storage processes

Corrective and Preventative Actions

Bioburden Chrom Viral Bioburden Chrom
Reduction Protein A Inactivation Reduction CEX • 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
Bioburden Chrom Virus Ultrafiltration Bioburden Bulk
Reduction AEX Filtration Diafiltration Reduction Storage and
Clearance Transport 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
 Case Study
Sporadic bioburden action-level excursions

Situation
• Sporadic mixed bioburden excursions at multiple
points in the downstream process

Root Cause
• Aseptic connections of equipment and sampling devices
Bioburden Chrom Viral Bioburden Chrom
Reduction Protein A Inactivation Reduction CEX

Corrective and Preventative Actions

• Short term:
• Retrained operators in aseptic techniques
• Long term:
• Reduced the number of aseptic connections

Bioburden Chrom Virus Bioburden Bulk

• Implemented sterile to sterile connectors and steam
Ultrafiltration
Reduction AEX Filtration Diafiltration Reduction Storage and to sterile connectors.
Clearance Transport
• Introduced a facility-wide sterile sampling system

Strategies to Address Bioburden Control in Downstream Processing

Case Study
Bulk solution contamination with Bacillus species

Situation
• Prefiltration bioburden load was 20 x the
specification
• Bacillus species suggested a steam-in-place issue

Root Cause Bioburden Chrom Viral Bioburden Chrom

Reduction Protein A Inactivation CEX
• Equipment design: improper pipe slope resulted in Reduction

cold spots that were insufficiently sterilized

Corrective and Preventative Actions

• Discard (scrap) the batch
• Redesign piping
• Requalify SIP (steam-in-place) cycle Bulk
Bioburden Chrom Virus Ultrafiltration Bioburden
Storage and
• New processes will use single-use storage systems Reduction AEX Filtration
Clearance
Diafiltration Reduction
Transport

Strategies to Address Bioburden Control in Downstream Processing

Key Points
Sources of bioburden

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

Strategies
to Address
Bioburden
Control in
Downstream
Processing
 3 Mitigation
Strategies

Strategies to Address Bioburden Control in Downstream Processing

Risk Mitigation Strategies

Assess

Mitigate

Monitor

Strategies to Address Bioburden Control in Downstream Processing

The risk profile varies from upstream to final fill
Risk Hammock

High

“The level of effort, formality and

documentation of the quality risk
management process should align
with the level of risk”
Risk

(ICH Q9A)

Aseptic Bioburden Controlled Aseptic

Low
Strategies
to Address Upstream Downstream Finish and Fill
Bioburden
Control in
Downstream
Operations
Processing
Assess Risk

Assess extent of risk, ability to detect,

Assess and frequency of occurrence

Mitigate

Monitor

Strategies to Address Bioburden Control in Downstream Processing

Microbiological Profile
Get to know the bioburden in your neighborhood

How many
of us? Will we cause
you
trouble?
Who are Where are
we? we
from?

Strategies to Address Bioburden Control in Downstream Processing

 Characterize Process Risk
Multiple tools are available

Fault Ishekawa
Statistical
Focus Objective Tree FMEA HACCP (Fish
Methods
Analysis Bone)
Risk Identification   
Risk
Risk Analysis  
Assessment
Risk Evaluation X  
Risk Reduction X   X

Risk Risk Acceptance X  X 

Control Risk Review X 
Risk Communication X X  

 Very suitable
Adapted from Roenninger S., Hertlein M. “Which Risk assessment fits
Strategies best?” Limited suitability
Logfile No 15 November 2011 1-4 Maas & Peither AG GMP Publishing
to Address
X Not suitable
Bioburden
Control in
Downstream
Processing
What parts of the process could
introduce bioburden?

(Hu)Man Mother Nature Material

Air Handling Water Tubing / Piping
Hygiene Insects
Experience Sanitizers Filters

Animals i.e. Rodents Disinfectants Resin

Aseptic Technique Microorganisms Buffers Tanks
Training
Humidity Storage Buffer Columns

Chromatography Mixing Transfer

Pump Calibration Flow Rate
Compounding Testing
Gaskets Transfer Panel
Bioburden profile Microscopy Enumeration
Water System Load cells Hold Time Sampling
Pressure Temperature
Steam Generator Clean room Room Cleaning
Weighing
Pump
Time Mass
Mixer Transfer
Filtration Data Acquisition Chromatogram
Air compressor Packing Installing

Machine Method Measurement

Strategies to Address Bioburden Control in Downstream Processing
How can I put the risks into
perspective?

Score
• Probability of occurrence
• Severity
• Ability to detect
• Criticality

Prioritize

CMC Biotech Working Group: A-Mab: A case Study in Bioprocess Development

Strategies to Address Bioburden Control in Downstream Processing

Assess Risks
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

Strategies to Address Bioburden Control in Downstream Processing

Mitigation Considerations

Assess

Eliminate source or reduce

Mitigate
likelihood of occurrence

Monitor

Strategies to Address Bioburden Control in Downstream Processing

Risk Mitigation
Mitigate
Material Considerations

Material Origin Bioburden profile

 Material consistency  What microorganisms are present?
 How many?
 Supplier transparency
 Variation over time?
 Quality management system
 Toxin producing?
 Quality philosophy  Spore formers?

Material Characteristics
 Growth Promoting
 Bacteriostatic
 Bactericidal

Strategies to Address Bioburden Control in Downstream Processing

Risk Mitigation Sanitization and Storage
Mitigate
Modes of action of sanitizers

Risk
Cell disruption results in byproducts
Sanitizer Modes of Action
Bacterial spores
• Spore coat penetration
Vegetative bacteria Reduce bioburden load
• Cell wall and cytoplasmic disruption Material selection
Fungi Containment
• Cell wall and cytoplasmic disruption • Single-use
Virus • Closed sampling
• Capsid and nucleic acid damage • Pre-packed columns
Removal
• Filtration

Strategies to Address Bioburden Control in Downstream Processing

Prevent Contamination by Containment
Mitigate
Implement Single-use Systems and Closed Sampling

Prevent
microbial
ingress

Eliminate
bioburden
contribution

Minimize
process
validation

Strategies to Address Bioburden Control in Downstream Processing

What is your objective?
Risk-based filter selection What filtration option is best
for my process?
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

Strategies to Address Bioburden Control in Downstream Processing

Downstream Filtration
Minimize Bioburden What filtration option is best for
my downstream process?

Bioburden reduction filters (0.45 or 0.2 m)

• May be sufficient for intermediate processing steps
• May be used as prefilters upstream of sterilizing- Goal: Minimize in-process bioburden
grade filters
• No requirement to validate sterility
• Manufacturers may validate bacterial reduction, but
not sterilizing performance • Reduce bioburden proliferation
• With other controls, bioburden reduction
filters may provide sufficient process safety
Sterilizing-grade rated filters (0.2 m)
• Prefiltration can increase filter capacity and
 Manufacturers must validate bacterial removal improve efficiency
 Traditionally used for intermediate processing steps

Strategies to Address Bioburden Control in Downstream Processing

Mitigate Risks
Mitigate
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

Strategies to Address Bioburden Control in Downstream Processing

Mitigation Strategy: Monitor

Assess

Mitigate

Monitor Monitor trends

Strategies to Address Bioburden Control in Downstream Processing

 B Bioburden
Downstream Monitoring
V Virus Monitor
What do I test for? Where? Why?
M Mycoplasma
E Endotoxin

B V B
E E E

Viral Bioburden Chromatography

Bioburden Chromatography
Inactivation Reduction CEX
Reduction Protein A

M
B
B V B
E
E E E

Bioburden Chromatography Virus Filtration Ultrafiltration / Bioburden Bulk Storage

Reduction AEX Clearance Diafiltration Reduction and Transport

Strategies to Address Bioburden Control in Downstream Processing

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

Key Points
Monitor
Mitigation Strategy

 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

Strategies to Address Bioburden Control in Downstream Processing

 Summary

Strategies to Address Bioburden Control in Downstream Processing

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

Strategies to address bioburden control in downstream processing
Recommended Reading

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
Acknowledgments
• Kerry Roche Lentine, Director, Technology Management, Global Growth Programs

Strategies to Address Bioburden Control in Downstream Processing

Somasundaram G.
Senior Technical Consultant

somasundaram.g@emdgroup.com

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