Addressing Downstream Challenges with Complex Injectables

The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Sterile filtration
of complex
injectables
Partha Banerjee
Senior Technology Consultant

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

Agenda
1
2
3
Background
Considerations for Sterile
filtration of liposomes
Liposome sterilization
methodology
4
5
Regulatory Guidelines
Essential parameters-
our observations
6
Sterile filtration of viscous
formulations
3
7
Sterile filtration of oils,
emulsions and ointments

• As per the US Food and Drug Administration (FDA), complex parenteral
products are those formulations which contain either complex ingredients or
API, complex formulation, i.e., delivery carrier, complex route of
administration, complex dosage form, or complex drug device combination
.
• The manufacturing of the complex injectable products is different and a great
level of observation of quality and care is required during their manufacturing,
packaging, distribution, and storage.
• Complex injectables have gained increasing attention due to their widespread
use in life-threatening and chronic diseases treatments. The category includes
diabetes, oncology, and hormonal therapy to name but a few.
Complex injectables – Background
Sterile Filtration of Complex Injectables
5

Complex injectables – what are they?
Complex drug products have become so prevalent that the FDA has defined them with the following
categories:
• Products with complex active ingredients (e.g., peptides, polymeric compounds, complex mixtures of
[active pharmaceutical ingredients]); complex formulations (e.g., liposomes, colloids); complex routes
of delivery (e.g., locally acting drugs, complex ophthalmological products and otic dosage forms that
are formulated as suspensions, emulsions, or gels); or complex dosage forms (e.g., implantables,
transdermals, metered dose inhalers, extended-release injectables
• Complex drug-device combination products (e.g., auto-injectors)
• Other products where complexity or uncertainty concerning the approval pathway or possible
alternative approach would benefit from early scientific engagement.
Complex processing challenges include, among others, aseptic manufacturing, the inclusion of highly
potent compounds, milling/particle engineering, spray drying, extrusion, and microfluidization.
Ref: ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin, Guidance for
Industry, FDA, May 2021.

Solubilization &
Bioavailability
Enhancement
Techniques
Drug-Eluting
Systems
The Four Categories of Complex Drug Products
In considering the above, complex drug products encompass a wide range of technologies and dosage
forms. However, we’ve found that these complex products generally revolve around four conversations
or categories:
Sterile complex
injectables
Highly Potent
APIs and/or
Controlled
Substances
7

• The FDA requires certain types of drug products to be
provided as a sterile dosage forms to avoid the possibility of
microbial degradation or infection occurring because of their
use.
• This includes several types of drug products,
including injectables (small or large volume parenteral
products), ophthalmic drugs, otic dosage forms,
and implantable products.
 Sterility of finished dosage forms can be assured via different
processes
Terminal Sterilization
Aseptic Manufacturing
Sterile Filtration
Sterile Complex Injectables
8

• Filtration is used for clarification purpose
(clarification filtration) and/or to sterilize solution
using sterilizing grade filter membranes (0.2 µ or
smaller pore size filters).
• Filtration of parenteral products ensures removal
of particulate matter and can be used either for
clarification or for sterilization purposes.
• As it’s a critical operation we classify filtration as
per
• Criticality and point of use.
• Usage.
Sterile filtration – Notable points
9

Filter in final fill – top 3 segmentations -
Utility filters
 Where process fluids come from
facility-wide systems, are not
tailored to a specific process and
do not have contact with the
drug substance or potential drug
substance.
 Part of a No-Impact System -
Where the equipment of system
has no impact, direct or indirect,
on product quality (ISPE
Commissioning & Qualification
Baseline Guide (2001))
 Filter does not affect product
quality (e.g. distribution gas
filter, water prefilter)
Critical
• Where process fluids “are in
direct contact with sterile final
product or critical surfaces of
the associated equipment.”
(PDA TR26)
• Part of Direct Impact System -
equipment or system that will
have focused and immediate
impact on product quality
(ISPE Commissioning &
Qualification Baseline Guide
(2001))
• Filter directly affects
product quality (e.g. sterile
hold vessel vent filter, sterile
liquid filter)
Moderately Critical
• Where process fluids “will
not be in direct contact with
exposed sterile product or
surfaces.” (PDA TR40)
• Part of an Indirect Impact
System - equipment or
system expected to have
incidental or secondary
impact on product quality
(ISPE Commissioning &
Qualification Baseline Guide
(2001))
• Filter indirectly affects
product quality (e.g. vent
filter in grade D, bioburden
reduction filter)
10

Filtration Portfolio – classified as per usage
Particulate Control Sterility Assurance
(LRV: 107 CFU/cm2)
Bioburden Control
(LRV: 106 CFU/cm2)
Milligard®
PolysepTM II
LifegardTM
Durapore®
(0.22 um)
Millipore
Express®
SHR
Millipore
Express®
SHF
Durapore®
(0.45 um)
Milligard®
PES
(0.2 um)
Milligard®
PES
(0.45)
Milligard ®
PES
(0.8 um)
Millipore
Express®
PHF
11

In this presentation we will be discussion the
sterile filtration and filter validation approach of
Liposomes, nano emulsions and viscous fluids.

Considerations for
Sterile filtration of
liposomes
13

Liposomes Characterization
 Size: small, intermediate, or large
 Number of lipid bilayers,
composition, and mechanism of
drug delivery
Small unilamellar vesicles (SUV)
− comprise a single lipid bilayer.
Diameter ~25 to 75 nm.
Large unilamellar vesicles (LUV)
− comprise of a single lipid bilayer.
Diameter >75 nm.
Multilamellar vesicles (MLVs)
comprise
− Contain many concentric lipid
bilayers. Diameter ~ 1-5 μm.
14

Preparation Methods for Liposomes
Three common approaches
Preparation of globules Size reduction Purification
The liposome preparation method affects purification!
15

PDI – Poly dispersity index
 PDI is basically a representation of distribution of size populations within a given sample
 Degree of non uniformity of size distribution particles
 Indicates a monodisperse system.
 PDI is a very essential parameter and its analysis helps to understand the size distribution of globule
based formulations.

Z Average Value
• The z-average is an intensity-based overall average size based on a specific fit to the raw correlation
function data. Basically the particle size.
Zeta Potential
• The zeta potential of a particle is the overall charge that the particle acquires in a particular medium.
Knowledge of the zeta potential of a liposome preparation can help to predict the fate of the liposomes
in vivo.
• Measurement of the zeta potential of samples is done using the technique of laser Doppler velocimetry
17

Liposome
sterilization
methods
18

Liposome Sterilization Methods: Advantages vs disadvantages
Ref: Liposomes as sterile preparations and limitations of sterilisation techniques in liposomal manufacturing ;April 2013;
Asian Journal of Pharmaceutical Sciences 8(2):88-95
19

Liposome Sterilization
Heat sterilization
• Not generally accepted
o Lipid/active not heat stable
o Leakage
o Safety issues
Gamma Irradiation
• Not generally used
• Degradation of lipid and
cholesterol
• Safety issues not assessed
• Cryo-radiation also not
effective
Sterile filtration
20

Liposome Sterilization by Filtration – the riddle
 Widely used
 Limited by vesicle size and size distribution
 Very little published information
 Challenges:
− allow particles (vesicles) of up to 300 nm to pass through
− retain bacteria that can be as small as 200 nm (width)
Lipid Globule B. Diminuta
21

Section 3 – Discussion on Liposome drug products – Guidance for Industry
Description of Manufacturing Process and Process Controls
We recommend including a detailed process flow diagram and a description of unit operations
with ranges for the process parameters and process controls.
These ranges should be supported by pharmaceutical development studies. The process and
mechanism of liposomal drug loading, as well as the removal of free (un-incorporated) drug
from the liposome formulation via purification should be described in detail. The
manufacturing process should be validated to demonstrate manufacturing process
consistency and reproducibility before commercial distribution.
Liposome drug products are sensitive to changes in the manufacturing conditions, including
changes in scale (size of the batches). Appropriate process controls should be established
during product development.
Prior knowledge can be leveraged and risk assessment techniques can be used to identify
manufacturing process parameters that potentially affect finished product quality.
Some examples of manufacturing process parameters that may affect liposome drug
performance are shear force, pressure, pH, temperature, batch-size-related hold times,
lyophilization parameters, etc. You should provide adequate justification for the selection of
the operating ranges for different batch sizes.
The physical and chemical complexity of liposome drug products present unique challenges to
the sterilizing filtration process. For example, components of liposomes could interact with the
filter matrix and clog it. Therefore, validated product-specific purification and sterilization
methods should demonstrate the ability of the microbial sterilizing filters to function correctly,
without compromising the integrity and structure of liposomes.
23
Ref: Liposome Drug Products, Guidance for Industry, FDA, April 2018

Regulatory Comments - Sterilizing Filtration of Liposomes
“Bulk holding times have been minimized or eliminated to control potential microbial
contamination. In response to a concern raised with respect to microbial contamination, a
pre-sterilized bioburden limit was adopted by the applicant above which batches will be
rejected.”
EMEA Scientific Discussion Documents
http://www.emea.eu.int
“The particle size distribution is measured as an important part of the in-process
controls.”
“…..followed by two 0.22 micron sterile filtration steps, aseptic filling, and
lyophilisation.”

Or – Can we follow draft guidance?
25
Ref: US Department of Health and Human Services, Food and Drug Administration, CDER: April 2018

We accept the potential challenges….Sterile filtration
• The sterility of such liposome solutions is typically ensured using
0.2μm rated sterilizing grade membranes, but due to the high
viscosity and low surface tension of these formulations, they can
cause pre-mature blocking and increased risk of bacterial
penetration through a 0.2μm sterilizing grade membrane.
• The low surface tension of liposome solutions affects the contact
angle with membrane and reduces bubble point leading to
bacterial penetration through the membrane.
• This poses a great challenge to select an appropriate
sterilizing grade membrane for a given process and for filter
manufacturers to develop a sterilizing grade membrane that
specifically addresses these needs.

Can I consider Aseptic Manufacturing?
Raw materials (including
organic and aqueous solvents,
the natural sources of lipid
components as well as other
additives such as buffers) are
sterilised after passing 200
nm filters.
The equipment can
be autoclaved and
sterilised.
Liposomes are
prepared and then
assembled into their
containers via
aseptic filling
Sources of contamination :
environmental air, operating personnel and the water for drainage) should be critically controlled by performing the filling process on work stations
in clean rooms.
Risk of contamination during aseptic processing remains
Especially if the initial raw materials are not sterilised adequately.
Limitations with natural sources of lipid components
Can only be subjected to filtration due to possible physicochemical degradation.
Contaminants
In the raw materials or introduced during manufacturing cannot be removed from the final product during aseptic
manufacturing is performed.
Terminal sterilization - active process of removing
Aseptic filling, and aseptic manufacturing - passive process of avoiding contamination

A typical Single use template for fill finish operations -

Essential
parameters-
our observations
29

Considering Particle size – Effect of Operating Parameters
31
Increased stirring rate -
• could improve droplet dispersion
• prevent droplet coalescence
• results in smaller apparent particle size
Optimum dispersion conditions -
• The dispersion condition of the droplets
changes according to its concentration in
the poor solvent.
• Therefore, the effects of feed rate of good
solvent and poor solvent ratio is important
• It controls the droplets concentration in
poor solvent, on the particle size of
nanospheres.
31

Considering elevated temperature of feed-
• At elevated temperature lipid membrane
passes from tightly ordered gel state (stable)
to a liquid crystal phase (metastable or
unstable)
• Freedom of movement of the individual
molecule is higher.
• This is due to the fatty acid chain adopting a
new conformation other than the all trans
state chain configuration, such as a gauche
confirmation state (chain tilt phenomena)

Considering sterile filtration - Case Study 1
Goals:
• Prefilter selection
• Understand the effect of high temp. and
pressure on the feed.
• Note – Particle size was below 150
micron but showed poor flow in SGF
Observations:
• Depth media works well compared to
membrane filters.
• High pressure may facilitate filtration,
but equally there is a risk of bleeding
and coagulation of particles.
• If particles coagulate, even taking the
feed temperature to phase transition
may not show any advantage in flow rate
through sterilizing grade filters.

FEED
Goals:
Sterile filtration operation.
• The PDI was 0.32, particle size was 84.3
nm, D90 was 1310 nm.
• Pressure applied was 15 PSI, feed
temperature equivalent to 60 degree C.
Observations:
• Its not always mean particle size, In the
sample analysis provided (Malvern particle
size analyser) the D90 value is 1310 (1.3
micron), this depicts that 90% of particles
are lesser than 1.3 micron and 10% more
than 1.3 micron.
• Similarly, the D90 value of the finished
product is 203 nm (0.203 micron) hence
both the solutions are difficult to filter by
0.2 um filter
• It is suggested that the particle size distribution should be
controlled in the liposome manufacturing stage.
• According to our experiences 150-200 nm samples can be
tried and closer the D90 value is to 200 nm lesser and lesser
is the throughput.
34

Goal:
 sterile filtration.
 The PDI was 0.15, particle size
was 90 nm, D90 was 160 nm.
 Pressure applied was 15 PSI,
feed temperature RT!!
 Observations:
 Depth media works well
compared to track itched
membrane.
 According to our experiences
150-200 nm samples can be
tried.
 The final filtration area was
significantly less and could be
replicated.

Considerations for
oils, emulsions and
ointments.
36

Sterile Filtration of Oils, Emulsions and ointments.
• Most pharmaceutical fluids are water based. But several hydrophobic APIs (Active Pharmaceutical
Ingredients) are often dissolved in an oily base, such as vitamins etc.
• Many of these components are heat sensitive and therefore sterile filtration is the most preferred way.
Examples of oily substances which are filtered are soy bean oil, castor oil, sesame oil, paraffin (liquid
and solid at ambient temperature), silicon oils etc.
• Examples for Emulsions used in the pharmaceutical industry are adjuvant solutions for vaccines or
Liposomes which are capable to solubilise hydrophobic APIs in a water environment. Other examples
include narcotics which is administered in soy bean oil in water emulsion with egg lecithin. Many
emulsions are non-Newtonian therefore flow over pressure curve is not linear.
• Some ointments can be heated up to more than 100°C but there are chances that the bacteria spores
can even survive. For that reason the preparation of oily pharmaceuticals got in the focus of the
regulatory bodies. By sterile filtration bacteria spores can be eliminated reliably.
37

High flow rates
Drying of the
filtration
equipment
Top points to consider while handling these formulation -
Product
specific
integrity
testing
Filter validation
aspects -
38

Optimizing the filtration train –
• Flow rate estimation prior to a filterability trial roughly can help. For this Darcy's Law utilised. Provided
we are working with a non Newtonian liquid. Very essential when we develop the formulation.
• This equation describes the flow of a fluid through a porous medium: Where Q is the flux or discharge
per unit area, e.g., m /s. Permeability of the medium, k (Sqm ) cross sectional area A (Sqm), and the
pressure drop (delta P), all divided by the dynamic viscosity, μ and the length the pressure drop is
taking place over.
• There are certain requirements for the sterilizing grade filtration of oils. Express range of filters are the
most suitable for filtration of oil containing liquids. So Polyethersulfone and Polyamide have good
chemical compatibility. Optimizing robust prefilters like Milligard PES (of varied pore sizes) can really
aid up the over process economics.
• Other important considerations include single use application in the process.

Filter validation aspects -
• Considering Validation aspects for sterile filtration
applications a bacteria challenge test. (BCT) has to
be performed. Prior to this test in a Viability study it
has to be demonstrated.
• Due to the high viscosity at ambient temperature of
different oily substances the filtration is performed at
60 to 80°C. At this temperature there is no viability
of the test bacteria given and we may need to
perform a two stage study.
• Unclear composition of different oils avoids a direct
detection in the contact solution specifically during
extraction and analysis.
• After filtration the oily liquid can not be removed by
water flushing from the membrane. For integrity
testing Product specific Integrity Test values can be
established for direct IT measurement after filtration.

Considerations for
viscous
formulations
41

Sterile Filtration of Viscous formulations -
• Viscosity enhancers are key ingredients in many lens care solutions and ophthalmic prescriptive drug
products.
• These additives are commonly cellulose based compounds but hyaluronic acid is becoming increasingly
popular in new formulations.
• Solutions containing viscosity enhancers can present difficulties during sterile filtration due to batch to
batch variability.
• Even with careful optimisation of the mixing process, premature filter blockage is still common
resulting in frequent filter changeouts mid-batch, product loss and increased processing time.
• And they pose challenges for sterile filtration.

• Multiple filter changeouts during batch processing: Premature filter clogging is evident even after
careful optimization of manufacturing process (eg Mixing) and filtration train.
• Product loss –
• The influence of raw materials and process parameters – Which may include granularity of these
cellulose based viscosity enhancers, Mixing techniques.
• Batch to batch variation.
• The temperature of the solution is also a very important factor during the filtration process, increasing
the temperature can promote gelation of the solution that will lead to premature clogging of a filter.
• What about binding of preservatives or essential API of the formulation to filter matrix – when flow rate
is less, contact time is on the higher sides.
• The filterability of solutions can change significantly depending on the time between mixing and
filtration.
• Sterile Filter validation.
Top points to consider while handling these formulation -

Optimizing the filtration train -
• Volume maximization study followed by a recheck during pilot scale runs –
• Advantages of composite asymmetric filter geometry
• Usage of a proper safety factor
To allow for process variability due to feed, process and membrane device in a robust process, a safety factor is typically included to define a
required filtration surface area. The required area for a process will be the minimum surface area for an average performance times the
safety factor. The actual variability of a process needs to be defined on a case-by-case basis. In absence of a detailed characterization study,
one could use the following typical economically rationalized safety factors for various unit operations as described in Herb Lutz, Journal of
Membrane Science 341 (2009) p268–278. Exact safety factors can be defined through experimentation. In case of anticipated large
variations (high relative standard deviation. RSD), safety factors more than recommended safety factor can be included in defining the
required surface area. In case specific information is available around low anticipated variability, a smaller safety factor can be used.
44

Approach 1 – Imitate process -
Complex
Injectables

Approach 2 – Pre-screening study (Filter validation)
Conducting the pre-validation screening study is not mandatory. But, if there is passage, it might be
easier to determine the mitigation plan prior to the retention validation instead of a retention test failure
investigation.
Assess – Process
duration, Actual
temperature, Actual
pressure, Actual
scale down volume
Important consideration –
Stable parameters.
Calculate – Scale down
volume, achieved per Square
surface area of the filter
Process contact time
Pre–screening to be conducted with
one filtration line only.
Pressure based study and no
recirculation mode.
Inoculation
47

Approach 3 –
Considering Standard vs High Area filtration devices.

Approach 4 – Selecting a well defined prefilter –
Like Milligard® PES
Benefits:
Fast flow and high throughput
Validated bioburden reduction (1.2/0.2 μm nominal and 1.2/0.45 μm pore sizes only)
Predictable scalability from small to production scale devices
High thermal stability: compatible with steam-in place and autoclave sterilization methods
Caustic stable
Gamma stable and available in single use assemblies

Points to consider in terms of
 Filterability
 Filter validation.
50

Filterability
Process considerations:
Keep vesicle size and size distribution small
Incorporate active in bilayer when feasible
Select process temperature in relation to Tc and lipid composition
Pre-wet filters with vehicle/buffer
Increase differential pressure gradually
May need to exceed certain differential pressure to initiate flow
Filter considerations:
Evaluate filterability early in process development
Evaluate different filter media types, hydrophilic PES generally works the best
Use pre-filtration to optimize filterability
51

Filter validation - Bacterial Retention study
Evaluate sterilization approach early in process development
Choose synthetic lipids when possible
Keep vesicle size and size distribution small
Evaluate different media pore size ratings and types
Consider “new technology” – Like stacked disc formats (Millipak® range of filters
with Durapore® membrane, high area device, AMPP (aseptic multi-purpose port)
Hydrophilic PES generally works the best

And we stand unique -
M LabTM /
Validation lab.
support
Liquid filters -
Durapore®
Liquid filters –
Millipore
Express ®
Gas filtration -
Aervent®
Next Gen
technology
Documentation
• Trusted name brand >40 yrs.
• Extreme strength
• Durable for reuse
• Low protein - preservative
binding
• High thermal and gamma
stability
• Fast flow
• Broad chemical compatibility
• Excellent wettability after
autoclaving
• High thermal and gamma stability
• From-buffer/media/protein
intermediates to viscous/complex
molecules – wide application.
• Meant for critical applications
• Sterility assurance
• Liquid bacterial retention
• Virus aerosol retention testing
• High air or gas flow rate
• Oxidation resistance for a long
service life
• Greater hydrophobicity
• Stacked disc membranes – for
higher flow/low hold up.
• AMPP – Aseptic multipurpose port –
protects product from
contamination, maintains sterility
• High area device - Unique shape,
taller pleats and narrower core –
double membrane area.
• Composite asymmetric membranes
– no its not 2 separate layers.
 Facilitating qualification
processes
 Supporting risk assessment,
management and mitigation
 Expediting approval
preparation and extending
compliance
• Want a pilot scale demo run.
• What IT troubleshooting – real
time view
• Visualize scale up scenario.
• Project status.
• Sample recon – if difficult to ship
• Ready to audit labs.

Product Support Services - Uniqueness continues -
Process Reviews
 Filter review
 Compliance review
 Integrity testing
review
Steam in Place review Training
• Filtration
• Integrity testing -Introductory
& Advanced Troubleshooting.
• IT testing - Compliance
• Filter validation
• SIP & Sterilization
• Microbiological analysis
• Sterility Testing
54

The vibrant M, Millipore®, M Lab™, Aervent®, Durapore®, Milligard®, Millipak®, Polysep®, Lifegard®, and Millipore Express® 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.
© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

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