Endotoxin Control Strategy in Biomanufacturing

The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
ENDOTOXIN CONTROL
STRATEGY IN
BIOMANUFACTURING
Somasundaram G (Som)
Senior Consultant, Asia Pacific
Global BioPharm CoE
Subhasis Banerjee Ph.D.
Principal Application Expert, APAC Bioprocessing
Customer Applications

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

Disclaimer:
Views expressed in this talk
constitute our professional opinion

Agenda
1
2
3
Endotoxin & sources of
contamination
Contamination Control
Strategy (CCS)
Raw material selection
4 Removal of endotoxin
5 Case Studies

Endotoxin &
sources of
contamination

What are Pyrogens?
Substances that induce fever when Ingested into the body by various routes
Origin/Source
Microbial - Bacteria
Non-microbial - Inorganic colloids, steroids
The most frequently encountered pyrogen in pharmaceutical processing is :
"Endotoxin" - material associated with the outer cell wall of gram negative
bacteria.
6
Endotoxin control strategy in biopharma manufacturing

 Endotoxins are continually shed from
bacteria during its life cycle; they are
also released when the bacterial cell
dies
7
Sources of Endotoxin Contamination
Image : wakopyrstar.com
 Probability of contamination exists
wherever bacteria are present - air,
water, food etc.

Physico - Chemical Characteristics of Endotoxins
Innermost region composed of lipid A
Hydrophobic
Responsible for pyrogenicity
Outer region composed of
polysaccharides
Hydrophilic
Structure
Composition
Complexes of Lipopolysaccharides (LPS)
containing lipids, carbohydrates and
proteins
Purified form (LPS) does not contain
protein
8

Physico-Chemical Characteristics of Endotoxins
Size
Their amphiphilic nature leads to aggregation in solution
Aggregates vary in size based on solution chemistry - 10,000-1000,000 Daltons has
been observed
Smallest irreducible size is found to be > 10,000 Daltons*
Charge
At pH > 2, endotoxins are negatively charged and behave as anions
The negative charge results due to the presence of acidic phosphate and carboxyl
groups in the molecule
* K. J. Sweadner et al., Applied and Environmental Microbiology, 34, 1977, p382.
9

Why Depyrogenate?
Prevention of endotoxin contamination, though ideal, cannot always be
ensured
Endotoxins are not removed during the sterilization step (sterile filtration or
autoclaving)
Sensitivity of mammals to Endotoxins is extremely high trace contamination
levels can induce severe reactions ranging fever to death –
(0.1 ng of LPS / kg body weight required to create an adverse reaction (1 EU/ml)
10

Assays for Endotoxin
(USP issue <23>, Chapter <151>, Chapter Endotoxin Test <85>)
Pyrogen Test
 Measures the temp. rise of rabbits following the intravenous injection of a test
solution
 Designed for products that can be tolerated by the test rabbit
 Dose: must not exceed 10 ml/kg injected; injection time not to exceed 10 minutes
Bacterial Endotoxin Test
 Uses a reagent Limulus Amebocyte Lysate (LAL) which reacts with endotoxins to form
a clot
 Endotoxin levels are measured by recording the spectrophotometric light absorbance
at suitable wavelengths
 Tests can be End-Point (gel clot) or kinetic (turbidimetric & colorimetric)
11

Contamination
control
strategy (CCS)

• Why & What is Contamination Control Strategy
• Understand the impact of bioburden excursions
• Recognize the sources of bioburden
• Develop strategies to mitigate risk
14
Contamination control strategy (CCS)

Regulatory Drivers
15
EU GMP Annex 1 Draft 2020:
‘A contamination control strategy should be implemented across the facility in order to assess the
effectiveness of all the control and monitoring measures employed’
CCS is mentioned 44 times in the update
• PIC/S GMP Part I, 2021:
‘Cross-contamination should be prevented for all products by appropriate design and operation of
manufacturing facilities. The measures to prevent cross contamination should be commensurate with the
risks. Quality Risk Management principles should be used to assess and control the risks’
‘5.10 At every stage of processing, materials and products should be protected from microbial and other
contamination’

16
Contamination Control Strategy: Holistic program
that encompasses concepts within the context of the
entire manufacturing facility and process
Facility Design: Designed according to GMP Regulations
/ Guidelines, Best practice and process requirements
Personnel: Gowning regime with personnel monitoring
program, suitable limits and action plan, training and
culture
Cleaning and Disinfection/Sterilisation: Consistent
removal of soiling and microbial load deactivation
Environmental Monitoring: Monitor viable and non-
viable, suitable limits and action plan
Process Simulation: End to end testing including
intervention assessment and worse case stressing
Process Design: Process controls including GMP and
GEP to ensure product is microbial/pyrogen, chemical,
non-viable and cross-contamination free.
What is a Contamination Control Strategy
Process
Design
Process
Simulation
Environmental
Monitoring
Cleaning &
Disinfection/Sterlisation
Personnel
Facility Design
Contamination
Control Strategy

Broad Range of Microorganism Retention
17
Minute virus of
mice (MVM)
Relevant
contaminant
Target organism
Typical LRV
above 4.0
Murine leukemia
virus (x-MuLV)
Model large virus
LRV > 6.1
B. diminuta
Standard model
bacteria
Tested by ASTM®
F838-05
LRV > 8
L. illini
Model spirochete
bacteria
Can penetrate
0.1 µm filters
LRV > 8
A. laidlawii
Model
mycoplasma
for 0.1 µm
filters
LRV > 8
M. orale
Relevant
contaminant
Can penetrate
0.1 µm filters
LRV > 8
VIRUS SPIROCHETE MYCOPLASMA BACTERIA

18
Microbial
Byproducts
Endotoxin
Exotoxin
Lipopeptides
Flagellin
DNA
Extracellular proteases
Bioburden
Bacteria
Fungi
Mycoplasma
Virus
TSE Agents
Why : Downstream bioburden and byproducts
Scope

Facility & Environment
Equipment
Processes
Materials
Utilities
Personnel
Each source
contributes
to the
process
bioburden
profile
Sources of bioburden
19
Staphylococcus
Bacillus
Non-fermenting
Gram Negative
rods
Aspergillus
Source: Public domain CDC/
Robert Simmons

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 can prevent many of these contaminations or excursions
Key Points
Sources of bioburden
20

Downstream
Risk profile & control strategies differ throughout the process
Secondary
Clarification
Chromatography
Protein A
Final Filling
Final 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
21

Assess
Mitigate
Monitor
Risk Mitigation Strategies
22

23
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

Control of Endotoxin Contamination: raw materials
• Inspection and Testing (LAL) of Contamination Sources:
Utilities
Water: WFI quality, Less than 0.25 EU/ml.
• Incoming Raw Materials
• In process Chemicals/Buffers
• Certificates
• Sampling etc.
• No beta-glucan interference for false-positive results
25

WFI specifications as per USP Chapter <1231>
https://www.pharmaguideline.com/2011/03/specification-for-water-for-injection.html
Sampling & testing of WFI
1. Description Clear, colorless & odorless liquid
2. Total Organic Carbon Note more than 500ppb
3. Conductivity (at 25’C) Less than 1.3mS/cm
4. Bacterial Endotoxins Less than 0.25EU/ml
5. Microbial limit test
a. Total aerobic microbial
count
b. Pathogens
i. Escherichia coli
ii. Salmonella sp.
iii. Pseudomonas aeruginosa
iv. Staphylococcus aureus
<10cfu/100ml
Should be absent
Should be absent
Should be absent
Should be absent
6. Acidity or alkalinity No red or blue color produced
7. pH Between 5.0 & 7.0
8. Ammonium <0.2ppm
9. Calcium and Magnesium A pure blue color is produced
10. Heavy metals <0.1ppm
11. Chlorides The solution shows no change in
appearance
12. Nitrates <0.2ppm
13. Sulphates The solution shows no change in
appearance
14. Oxidizable substances The solution remains faintly pink
15. Residue on evaporation <0.001%
https://www.sigmaaldrich.com/IN/en/product/mm/pyr0matkit

Raw materials selection : COQ
27
Cell culture media (Add BPM
product COA)
Sterilizing grade filter

28
1X0 devices flushed with water/buffer, as indicated (600 LMH, 50 L/m2
or 25 L/m2 with buffer)
Format
Beta-glucan LAL assay
(pg/mL)1
water buffer
X0HC < 25.3 < 80
X0SP < LOQ < LOQ
β-(1-3) Glucan Significance
▪ Operational
β-Glucan can cause false positive in traditional
Limulus Amebocyte Lysate (LAL) test for
detecting endotoxins.
▪ Regulatory
β-Glucans are considered as “process-related
impurities” categorized under downstream derived
impurities as per International Conference on
Harmonization (ICH) Topic Q6B.
▪ Patient Safety
β-Glucan levels in patient’s blood streams are
monitored for detection of fungal and bacterial
infections.
• No beta-glucans to interfere with limulus
amoebocyte lysate (LAL) testing for bacterial
endotoxins
Raw materials selection : beta-glucan

Methods of Endotoxin Removal
Non-Filtration
Depyrogenation achieved by inactivation of the molecule
Examples of Processes: Acid/Base or Polymyxn B Treatment, Hydrogen
Peroxide (Oxidation), Heat (Moist and Dry)
Pyrogenicity of the molecule is 'lost' by structural changes; 'the altered
molecule' may still be present
Filtration:
Depyrogenation achieved by removal of the molecule
Examples of Processes:
Size Exclusion - Separation based on size (RO &UF)
Adsorption - Separation based on charge or hydrophobic interaction (MF or
depth filtration
AEX chromatography
Binding and elute chromatography
Depends on the isoelectric point of the protein
pH range stability of the protein
30

Endotoxin Removal by Filtration:
Size Exclusion
Principle - effective 'pore' size of the membrane filter is smaller
than the effective size of the endotoxin
Membrane "Pore" Rating - membranes are available in a
range of pore sizes that have a correspondence to the MWs of
marker proteins they can retain - classified into ultrafiltration,
nanofiltration & reverse Osmosis
Membrane Mol. Weight Cut Off (kilodaltons)
100
kD
1000
kD
0.1
kD
1
kD
10
kD
Membrane Process
Reverse
Osmosis
Nanofiltrati
on
Ultrafiltration
"Loose"
"Tight"
31

Size Exclusion - Endotoxin Aggregation State
Solution
Endotoxin
Aggregation Form
Size
Water Vesicle Submicron
Saline 0.9% Vesicle Submicron
MgCl2 5 mM Vesicle Submicron
EDTA 5 mM Micelle 300-1000 kD
1% Sodium deoxycholate
Micelle or Sub Unit
down to 10-20 k
Daltons
2% Sodium deoxycholate
& 5mM EDTA (Worst
Case)
Sub unit 10-20 k Daltons
K. J. Sweadner et al., Applied and Environmental Microbiology, 34, 1977, p382.
Endotoxin Size Depends on Solution Chemistry!
How big is the endotoxin molecule?
32

Size Exclusion - membrane selection
Membrane Flux
LRV
100 kD 1000 kD
1 kD 10 kD
Endotoxin Aggregate
Range
Soln. 1
Soln. 2
Aggregation State of Endotoxin -
high LRV
High Process Flux - Affects
System Size
Ease of passage of Other Solutes -
buffer salts, sugars, amino acids
etc.
Membrane/solvent compatibility
33

Endotoxin Removal by Filtration
Size Exclusion - what do you choose?
Depends on knowledge of endotoxin aggregation size & extent of
LRV desired - pilot testing at worst case conditions.
Classically, ultrafiltration using a 10 kD membrane is a widely
employed process.
A more open ultrafiltration may give the benefits of higher flux -
leads to lower system size/cost
34

A 100 kD UF membrane may also be used to remove endotoxins if they
are aggregated to a size >> 100 kD
Nanofiltration (0.1-1 kD) may be used for endotoxin removal from
solutions with very low MW solutes (NaCl, amino acids) & higher
filtrate purity.
Reverse Osmosis (< 0.1kD) is good only for removal of endotoxins
from water
35
Size Exclusion – additional comments

Ultrafiltration (TFF):
Effectiveness in Depyrogenation
Solution 0.22 m 0.025 m
1,000 kD
NMWCO
100 kD
NMWCO
10 kD
NMWCO
Water 0 > 4 > 4 > 4 > 4
0.9% NaCl 0 > 4 > 4 > 4 > 4
5 mM MgCl 0 > 4 > 4 > 4
5 mM EDTA >= 4 > 4 > 4
1% Na Cholate 1 2 > 5
2% Na Cholate
& 5 mM EDTA
0 0 > 5
1% Na
Deoxycholate
0 0 > 5
A Range UF Membranes (10 - 1000 kD) can retain Endotoxins depending on Solution Condition
(LRV on Membranes)
36
Endotoxin control strategy in biopharma manufacturing Internal data

Adsorptive Forces
Electrostatic - Due to charge/charge interaction; Unlike charges attract and
like charges repel
Dispersion or Van der Waals - Forces that exist between all atoms &
molecules; quantum mechanical in origin; generally weak
Hydrogen Bonding - Ability of a H atom bound to electronegative atom (O)
to attract other electronegative atoms; unique electrostatic attraction
Hydrophobic interactions - Entropic in origin; results from the need for
water molecules to maximize their conformational disorder.
37

Endotoxin Adsorption: Results
Membrane
Liquid
Filtered
EU/cm2 LRV
Charged Durapore®,
0.2 micron
Water > 5 x 105 > 5.0
Charged Durapore®,
0.2 micron
Mannitol > 400 >1.5
Charged Durapore®,
0.2 micron
Water with
150 mM NaCl
very low very low
Hydrophobic
Durapore®, 0.2 micron
Water with
150 mM NaCl
>1630 > 3.35
Hydrophobic
Durapore®, 0.45
micron
Water with
150 mM NaCl
1630 3.1
38

Limitations of Adsorption Processes
Adsorption processes (charged & hydrophobic) can proceed only
until the membrane capacity is not exhausted
when adsorption sites are filled, breakthrough occurs
In general, adsorption processes depend on solution chemistry
pH, Ionic Strength, competitive adsorption by other solutes
39

40
Endotoxin Removal by AEX chromatography
Anion
exchange
High ionic
strength
Low ionic
strength
Non-binding sample constituents
Chromatogram

o Endotoxin pI 2-4
o For basic proteins pI > 7 , ET binds to AEX matrix at neutral pH eluting at a
higher conductivity than proteins
o For acidic proteins : ET may co-adsorbed along resulting in product loss
o Use of detergents in the wash step facilitates ET removal by chrome
41
Endotoxin Removal by AEX chromatography

Clearance vs. Yield
Anion exchange chromatography Bind & Elute separation
Both AEX resins show similar performance:
• 2.5 – 3.0 LRV endotoxin clearance
• 85 – 90% BSA yield
Feed: Endotoxin 5.0E+05 EU/mL + BSA 5 mg/mL
Fractogel® DEAE & Eshmuno® Q at 3.4mL CV (10cm BH)
the elution pool was analyzed for
endotoxin LRV and BSA yield.
E.Coli endotoxin (pI ~2) and BSA(pI ~4.8) as
model protein spiked into buffer.
Loading feed was formulated at pH5 and no
salt for both binding.

Clearance vs. Yield
Anion exchange chromatography Flow-Through separation
Fractogel® DEAE shows better performance:
• Endotoxin clearance 1.4 LRV at DEAE
and 0.9LRV at Q
• ≥ 87% flowthrough of BSA
Feed: Endotoxin 5.0E+05 EU/mL + BSA 5 mg/mL
Fractogel® DEAE & Eshmuno® Q at 3.4mL CV (10cm BH)
the elution pool was analyzed for
endotoxin LRV and BSA yield.
E.Coli endotoxin (pI ~2) and BSA(pI ~4.8) as
model protein spiked into buffer.
Loading feed was formulated at pH5 with 8-10
mS/cm sodium chloride.

Case Study
Bioburden/Endotoxin 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

Case Study
Bioburden action-level excursions of 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
• 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.

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
• 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

48
Conclusion
• Endotoxins have been & going to be one of the challenges in Biopharma manufacturing.
• If not controlled, they could pose serious consequences to the manufacturer & finally to the
patient.
• As described, endotoxin control should be ensured for the safety of the drug
• Selection of Raw materials with low endotoxin levels should be used/preferred
• Bioburden control & monitoring plays a major role in reducing endotoxins in the
process/product
• Downstream Unit operations should be optimized for robust control of Endotoxins

© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. The vibrant M, Millipore®
Durapore®, Fractogel® and Eshmuno® 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.
THANK YOU
Somasundaram G. Dr. Subhasis Banerjee

Endotoxin Control Strategy in Biomanufacturing

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