ASTM E3409-24

• Abstract:

  SIGNIFICANCE AND USE; 5.1 Liposomal formulations for the treatment of cancer and other diseases are the most common form of nanotechnology-enabled drug products submitted for market approval and in clinical application at the present time. The accurate characterization of their physical-chemical properties is critical to support the development and assessment of such products (2). In particular, size, size distribution, shape, and physical stability are key properties (among others) that have been widely identified as critical quality attributes (CQAs) for liposomal drug products. ; 5.1.1 Asymmetrical-flow field-flow fractionation (AF4) is a chromatographic-like technique that uses hydrodynamic forces to gently separate analytes into their component populations according to size and diffusivity (3 and 4). The fractionated sample then passes through one or more online detectors chosen specifically for the application requirements. The combination of low-shear separation, tolerance for complex matrices, and exceptionally broad size range make AF4 a technique of choice for application to nanotechnology-enabled drug products such as liposomes (13, 5-9). ; 5.1.2 Multi-detector AF4 is suitable for research and development, manufacturing quality control, product stability/shelf-life testing and regulatory assessments. ; 5.1.3 There are multiple assumptions inherent in the application of MD-AF4, including the appropriateness of models used to interpret online light scattering data and the compatibility of the analyte and matrix with the membrane that forms the surface of the accumulation wall in the fractionation channel. Other assumptions are detector or analyte specific. ; 5.2 Chemical components of the mobile phase must not induce agglomeration of liposomes or otherwise significantly alter their physical properties. ; 5.3 Discretion should be used in the interpretation of size data obtained by different scattering detectors using different scattering models and modes of analysis. ; 5.4 ...; SCOPE; 1.1 This test method describes a measurement procedure to reproducibly separate component size populations present within liposomal drug formulations and to characterize their associated size and size distribution. The method can also yield information on the shape and physical stability of the liposomes and is applicable to measurements in the presence of serum proteins. Fractions can be collected for off-line analysis using various techniques not specified in this test method. ; 1.2 This test method applies to uni-lamellar and multi-lamellar liposomes that are designed for drug delivery and which are dispersed in a native solution that is aqueous in nature. The method is generally applicable over a particle size range (radius) of approximately 10 nm to 250 nm, and for injected lipid mass from 20 µg to 200 µg. ; 1.3 This test method is based on the multi-detector asymmetrical-flow field-flow fractionation (MD-AF4) technique as configured on a typical commercial instrument platform with online detectors such as multi-angle (static) light scattering (MALS), dynamic light scattering (DLS), ultraviolet-visible (UV-Vis) absorbance, and differential refractive index (dRI) (1).2 ; 1.4 This method does not address liposome composition. Refer to Test Methods E3297, E3323, or E3324 for lipid quantification. ; 1.5 Units—The values stated in SI units are to be regarded as standard. Where appropriate, cgs units are given in addition to SI. ; 1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. ; 1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of In...

• Status: Published
• Publication Date: 31-Jan-2024
• Technical Committee: E56 - Nanotechnology
• Drafting Committee: E56.02 - Physical and Chemical Characterization

• Refers: ASTM E1617-09(2024) - Standard Practice for Reporting Particle Size Characterization Data
• Effective Date: 01-Feb-2024
• Refers: ASTM E1617-09(2019) - Standard Practice for Reporting Particle Size Characterization Data
• Effective Date: 01-Apr-2019

Overview

ASTM E3409-24: Standard Test Method for Analysis of Liposomal Drug Formulations Using Multidetector Asymmetrical-Flow Field-Flow Fractionation (MD-AF4) establishes a standardized procedure for the physical characterization of liposomal drug formulations. Liposomes are widely used as nanotechnology-enabled drug delivery systems, especially for treating cancer and other diseases. Accurate assessment of their size, size distribution, shape, and physical stability is essential for regulatory approval, quality assurance, and effective pharmaceutical development.

This method uses multidetector asymmetrical-flow field-flow fractionation to separate and analyze liposome size populations and their characteristics. The approach supports reproducible, high-resolution measurements and is adaptable for both unilamellar and multilamellar liposome products.

Key Topics

• Liposomal Drug Characterization: Focuses on critical quality attributes (CQAs) such as particle size, size distribution, shape, and physical stability.
• MD-AF4 Principle: Asymmetrical-flow field-flow fractionation is a low-shear, gentle technique that separates analytes based on size and diffusivity, making it ideal for sensitive liposomal formulations.
• Multidetector Setup: Supports integration with online detectors, including multi-angle light scattering (MALS), dynamic light scattering (DLS), UV-Vis absorbance, and differential refractive index (dRI), ensuring comprehensive data collection.
• Compatibility and Assumptions: Stresses the importance of the compatibility between samples/matrices and the fractionation membrane, and the need for proper interpretation of light scattering data using appropriate models.

Applications

The ASTM E3409-24 standard is designed for a range of practical uses in the pharmaceutical and biotechnology industries:

• Research and Development: Enables detailed studies of new liposomal formulations, supporting innovation in drug delivery.
• Quality Control in Manufacturing: Assures consistent production of liposomal drug products by monitoring and controlling key physical-chemical attributes.
• Product Stability and Shelf-Life Testing: Assists in evaluating changes in liposome characteristics under various storage conditions to predict product performance over time.
• Regulatory Compliance: Provides a standardized and validated analytical approach to support submissions to regulatory agencies for market approval.
• Comparative Assessment: Offers a high-resolution fractionation alternative to traditional dynamic light scattering (DLS) for more accurate analysis of heterogeneous populations.

Scope limitations:

• This method focuses on the physical characterization (size, shape, stability) and does not address liposome composition-separate standards cover lipid quantification.
• It applies to both uni-lamellar and multi-lamellar liposomes in aqueous environments and a typical particle size range from approximately 10 nm to 250 nm.

Related Standards

ASTM E3409-24 should be used in conjunction with other relevant international and ASTM standards for full characterization and quantification of liposomal formulations:

• ASTM E3297: Lipid Quantification by HPLC with Charged Aerosol Detector (CAD)
• ASTM E3323: Lipid Quantification by HPLC with Evaporative Light-Scattering Detector (ELSD)
• ASTM E3324: Lipid Quantification by UHPLC with Triple Quadrupole Mass Spectrometry (TQMS)
• ASTM E3247: Nanoparticle Size Measurement in Aqueous Media by DLS
• ISO/TS 21362: Nanotechnologies-Analysis of Nano-Objects Using AF4
• ISO 22412: Particle Size Analysis-Dynamic Light Scattering

Practical Value

By adopting ASTM E3409-24, laboratories and manufacturers achieve:

• Reproducibility and Objectivity: Well-documented, consensus-based procedures ensure consistent, repeatable analysis critical for regulatory submission and routine QC.
• Enhanced Data Quality: Multi-detector AF4 offers superior resolution and insight into complex or polydisperse liposome populations, reducing risk of misleading data.
• Regulatory Confidence: Compliance with internationally recognized standardization principles supports clear communication with authorities and global market access.

This standard is a key resource for ensuring the safe, effective, and reliable development and control of liposomal drug products in modern nanomedicine.

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3409 − 24
Standard Test Method for
Analysis of Liposomal Drug Formulations Using
Multidetector Asymmetrical-Flow Field-Flow Fractionation
This standard is issued under the fixed designation E3409; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.7 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.1 This test method describes a measurement procedure to
ization established in the Decision on Principles for the
reproducibly separate component size populations present
Development of International Standards, Guides and Recom-
within liposomal drug formulations and to characterize their
mendations issued by the World Trade Organization Technical
associated size and size distribution. The method can also yield
Barriers to Trade (TBT) Committee.
information on the shape and physical stability of the lipo-
somes and is applicable to measurements in the presence of
2. Referenced Documents
serum proteins. Fractions can be collected for off-line analysis
2.1 ASTM Standards:
using various techniques not specified in this test method.
D1193 Specification for Reagent Water
1.2 This test method applies to uni-lamellar and multi-
E1617 Practice for Reporting Particle Size Characterization
lamellar liposomes that are designed for drug delivery and
Data
which are dispersed in a native solution that is aqueous in
E3144 Guide for Reporting the Physical and Chemical
nature. The method is generally applicable over a particle size
Characteristics of Nano-Objects
range (radius) of approximately 10 nm to 250 nm, and for
E3206 Guide for Reporting the Physical and Chemical
injected lipid mass from 20 μg to 200 μg.
Characteristics of a Collection of Nano-Objects
1.3 This test method is based on the multi-detector
E3247 Test Method for Measuring the Size of Nanoparticles
asymmetrical-flow field-flow fractionation (MD-AF4) tech-
in Aqueous Media Using Dynamic Light Scattering
nique as configured on a typical commercial instrument plat-
E3297 Test Method for Lipid Quantitation in Liposomal
form with online detectors such as multi-angle (static) light
Formulations Using High Performance Liquid Chroma-
scattering (MALS), dynamic light scattering (DLS),
tography (HPLC) with a Charged Aerosol Detector (CAD)
ultraviolet-visible (UV-Vis) absorbance, and differential refrac-
E3323 Test Method for Lipid Quantitation in Liposomal
tive index (dRI) (1).
Formulations Using High Performance Liquid Chroma-
tography (HPLC) with an Evaporative Light-Scattering
1.4 This method does not address liposome composition.
Detector (ELSD)
Refer to Test Methods E3297, E3323, or E3324 for lipid
E3324 Test Method for Lipid Quantitation in Liposomal
quantification.
Formulations Using Ultra-High-Performance Liquid
1.5 Units—The values stated in SI units are to be regarded
Chromatography (UHPLC) with Triple Quadrupole Mass
as standard. Where appropriate, cgs units are given in addition
Spectrometry (TQMS)
to SI.
2.2 ISO Standards:
1.6 This standard does not purport to address all of the
ISO/TS 21362 Nanotechnologies—Analysis of Nano-
safety concerns, if any, associated with its use. It is the
Objects Using Asymmetrical-Flow and Centrifugal Field-
responsibility of the user of this standard to establish appro-
Flow Fractionation
priate safety, health, and environmental practices and deter-
ISO 22412 Particle Size Analysis—Dynamic Light Scatter-
mine the applicability of regulatory limitations prior to use.
ing (DLS)
1 3
This test method is under the jurisdiction of ASTM Committee E56 on For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Nanotechnology and is the direct responsibility of Subcommittee E56.02 on contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Physical and Chemical Characterization. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved Feb. 1, 2024. Published February 2024. DOI: 10.1520/ the ASTM website.
E3409-24. Available from International Organization for Standardization (ISO), ISO
The boldface numbers in parentheses refer to a list of references at the end of Central Secretariat, Chemin de Blandonnet 8, CP 401, 1214 Vernier, Geneva,
this standard. Switzerland, https://www.iso.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3409 − 24
2.3 Code of Federal Regulations: 3.1.9 detector flow, n—in field-flow fractionation, that por-
21 CFR § 211.194(a)(2) FDA Current Good Manufacturing tion of mobile phase that exits the channel and enters the
Practice for Finished Pharmaceuticals detectors. Adapted from ISO/TS 21362
3.1.9.1 Discussion—While typically detector flow and chan-
3. Terminology
nel flow are identical, in some configurations channel flow can
be split before entering the detectors, for example, to concen-
3.1 Definitions:
trate the analyte by purging a portion of the analyte-free mobile
3.1.1 accumulation wall, n—surface of a field-flow fraction-
phase.
ation channel toward which sample components are driven by
the applied field acting perpendicular to the channel flow.
3.1.10 elution, n—in field-flow fractionation, a process by
ISO/TS 21362
which analytes in the mobile phase, or eluent, are transported
3.1.1.1 Discussion—In asymmetrical-flow field-flow
through, and exit from, the fractionation channel. ISO/TS
fractionation, the accumulation wall is flat and consists of a
replaceable semipermeable membrane on a porous frit sub-
3.1.11 elution time, t, n—in field-flow fractionation, the
strate.
elapsed time beginning with the initiation of elution following
3.1.2 asymmetrical-flow field-flow fractionation (AF4),
sample injection and excluding pre-elution steps such as
n—separation technique that uses a cross flow field applied
focusing, relaxation or transitions.
perpendicular to the channel flow to achieve separation based
3.1.11.1 Discussion—Retention and elution share an equiva-
principally on analyte diffusion coefficient or size. Adapted
lent timeline and may be used interchangeably. However,
from ISO/TS 21362
retention is principally used in reference to specific retained
species (for example, an analyte) eluting more slowly than the
3.1.3 band broadening, v—overall dispersion or widening of
unretained tracer (that is, the void peak) due to their specific
an analyte band as the analyte passes through a separation
properties and interaction with the applied field. A graphical
system. ISO/TS 21362
representation of a fractogram generally shows elution time or
3.1.4 channel, n—in field-flow fractionation, a thin ribbon-
retention time on the (horizontal) x-axis, after the time asso-
like chamber with a parabolic flow profile required for sepa-
ciated with pre-elution steps has been subtracted.
ration under the influence of a field applied perpendicular to the
3.1.12 field-flow fractionation, n—separation technique
channel flow. ISO/TS 21362
where a field is applied to a liquid passing along a narrow
3.1.5 channel flow,, n—in field-flow fractionation, the total
channel to induce separation of analytes present in the liquid,
parabolic laminar-flow of eluent or mobile phase through the
dependent on their differing mobility under the force exerted
channel. Adapted from ISO/TS 21362
by the field. Adapted from ISO/TS 21362
3.1.5.1 Discussion—For purposes of this test method, chan-
3.1.13 focusing, v—in asymmetrical-flow field-flow
nel flow and detector flow are equivalent. If the operator uses
fractionation, a process by which, during and after sample
a post-fractionation split-flow device to divert part of the
injection, a counter-balanced flow entering from opposite
channel flow to waste (for example, to concentrate the analyte),
directions in the channel is applied to focus the sample
then the detector flow will be less than the channel flow.
components into a thin band close to the inlet port and near the
3.1.6 channel spacer, n—in asymmetrical-flow field-flow
accumulation wall. Adapted from ISO/TS 21362
fractionation, a thin plastic film with a cut-out that defines the
3.1.13.1 Discussion—While conventional channels require
nominal thickness and lateral dimensions of a channel.
a focusing step, there are channel designs, such as the frit-inlet
Adapted from ISO/TS 21362
channel, that do not require focusing. Focusing is also associ-
3.1.6.1 Discussion—The geometry of the channel is typi-
ated with the process of sample relaxation.
cally defined by a trapezoid shape with breadth typically ca.
20 mm to 25 mm, length typically ca. 100 mm to 300 mm and 3.1.14 fractogram, n—in field-flow fractionation, a two-
dimensional graphical representation of data derived from an
height typically between 190 μm and 500 μm. The nominal
channel height and lateral dimensions can also be defined using experiment, typically with one or more detector signals on the
vertical y-axis and time on the horizontal x-axis. Adapted
fixed-height channels that do not require a spacer.
from ISO/TS 21362
3.1.7 channel thickness (height), w, n—in field-flow
3.1.15 hydrodynamic radius, R , n—the sphere-equivalent
fractionation, the nominal vertical separation between the
o
depletion wall and the accumulation wall. ensemble average radius that reflects the central tendency of
3.1.7.1 Discussion—The effective thickness can vary de- the underlying population of particles as determined by dy-
pending on the membrane used and the degree of compression namic light scattering. Adapted from Test Method E3247
of the membrane as well as other factors.
3.1.15.1 Discussion—The hydrodynamic radius can be ob-
tained from different computational methods, including, for
3.1.8 cross flow, n—in asymmetrical-flow field-flow
instance, the cumulants method combined with the Stokes-
fractionation, the flow field applied perpendicular to the
Einstein equation. Note that when obtained from cumulants the
channel flow to achieve separation of analytes. ISO/TS 21362
average hydrodynamic radius is often referred to in the
literature as the z-average (z-avg) size, a legacy term. This
measurand can be scattering-angle-dependent and can be
Available from U.S. Government Publishing Office (GPO), 732 N. Capitol St.,
NW, Washington, DC 20401, http://www.gpo.gov. influenced by other factors besides size.
E3409 − 24
3.1.16 injection flow, n—in field-flow fractionation, flow 3.1.23.1 Discussion—Typically expressed as the ratio of the
that drives the sample out of the injection loop and into the void peak elution time to the analyte peak retention time. For
fractionation channel. ISO/TS 21362 efficient separation, R should be less than 0.2.
3.1.17 liposomal formulation, n—product designed to assist
3.1.24 retention time, t , n—in field-flow fractionation, the
R
in the delivery of an active pharmaceutical ingredient either
time between initiation of fractionation and detection of an
encapsulated or intercalated in the liposome.
analyte peak defined at its maximum signal intensity. ISO/TS
3.1.17.1 Discussion—Water-soluble drugs are contained in
the aqueous compartment and hydrophobic drugs are contained
3.1.24.1 Discussion—Retention time for an analyte is gen-
within the lipid bilayer. Additional molecular species are
erally determined from the mass concentration detector signal
incorporated into the bilayer to control functionality (for
at an elution time associated with the fractionated analyte peak.
example, distearoyl phosphatidylethanolamine-PEG2000-
Retention time and elution time share the same effective
amine is commonly used to confer colloidal stability and
timeline. In principle, retention time can be assigned based on
minimize non-specific interactions). The finished drug product
any signal that tracks the elution of the analyte. Note that
might also contain inactive ingredients (excipients) in addition
retention time for many colloidal particles, including
to the lipids and active pharmaceutical ingredient(s).
liposomes, can differ between mass concentration and light
3.1.18 liposome, n—synthetic vesicle composed of one or
scattering detectors due to differences in detector sensitivity
more bilayers formed by amphipathic molecules such as toward analyte size.
phospholipids that enclose one or more aqueous compartments
3.1.25 root mean square radius (radius of gyration), R ,
g
and hydrophobic regions.
n—in multi-angle static light scattering, the root mean square
3.1.19 mobile phase (eluent), n—liquid phase used to
distance of a scattering object’s component parts from its
achieve transport and separation of analytes in field-flow
center of mass.
fractionation or chromatography.
3.1.25.1 Discussion—In a light scattering measurement,
3.1.20 recovery, n—in field-flow fractionation, ratio of the
object refers to a microscopic particle dispersed in a liquid that
analyte mass eluted during fractionation to the initial injected
scatters light with a differential angular distribution of inten-
mass, expressed as a percentage. ISO/TS 21362 sity.
3.1.20.1 Discussion—In practice, recovery is determined
3.1.26 selectivity, S, n—in field-flow fractionation, a mea-
using an appropriate online mass concentration detector by
sure of fractionation power related to the change in retention
comparing the area under the mass concentration trace ob-
time relative to the corresponding change in particle size.
tained with and without application of the applied field and
3.1.26.1 Discussion—Selectivity can be determined experi-
focusing. Recovery is considered acceptable if at or above
mentally from the slope of log t versus log 2r, where r is
R
70 % according to ISO/TS 21362; higher recoveries are antici-
radius measured by MALS or DLS. These measurements are
pated for high quality (for example, clinical) liposomal drug
typically performed under conditions similar to those used for
formulations. In this test method, recovery includes all mass
sample analysis, but with materials of known size (for
eluted during the elution program (post void peak), including
example, PSL spheres). For AF4, S can exceed unity, where the
any residual material released after cessation of cross flow
higher the value of S, the better the resolving power.
(report this value as “total recovery”). The user may also
choose to report a recovery value that excludes residual d log t
R
S 5 (1)
U U
d log 2r
material (that is, material eluting after cross flow is removed).
In this case, the recovery value should be reported as “field-on
3.1.27 void peak, n—in field-flow fractionation, a peak
recovery.”
appearing early in the fractogram that corresponds to
3.1.21 relaxation, v—in field-flow fractionation, process by
unretained, typically small sample components that are not in
which the sample components assume their equilibrium state
equilibrium with the separation field. ISO/TS 21362
with respect to the opposing forces of diffusion and the applied
3.1.27.1 Discussion—The void peak, typically very small
field before elution is initiated. ISO/TS 21362
and narrow, is used to define the void time t . The void peak is
3.1.22 residual peak, n—in field-flow fractionation, a peak always present as an artifact of the hardware change from focus
mode to elution mode, and elutes at the average velocity of the
appearing in the fractogram after the applied field is removed
(field-off) and which corresponds to unfractionated material channel flow. An uncharacteristically large void peak indicates
substantial unretained material is present, suggesting the elu-
retained on the accumulation wall during application of the
applied field. tion conditions might not be acceptable for that sample; further
method development might be necessary to resolve this issue,
3.1.22.1 Discussion—This “peak” often contains particles
or fragments that are too large to migrate under the applied or the sample might not be appropriate for AF4 analysis.
field or which for some other reason strongly interact with the
3.1.28 void time, t , n—in field-flow fractionation, the elu-
membrane or channel surface. The peak is frequently ill-
tion time associated with the maximum of the void peak.
defined (that is, atypical shape).
3.2 Acronyms and Symbols:
3.1.23 retention ratio, R, n—in field-flow fractionation, the
3.2.1 AF4—asymmetrical-flow field-flow fractionation
ratio of the velocity of the analyte to the average velocity of the
fluid in the channel. 3.2.2 BSA—bovine serum albumin
E3409 − 24
3.2.3 CF—channel flow—flow exiting the channel; equiva- instrument settings (for example, MALS angles, DLS angle,
lent to DF in this method detector flow rate, cell configuration) allow for accurate
analysis of particles in the size range of interest for application
3.2.4 DAD—diode array detector (UV-Vis absorbance)
of the test method.
3.2.5 DF—detector flow; equivalent to CF in this test
4.5 Calibration/normalization of the MALS detector is per-
method
formed periodically according to manufacturer instructions.
3.2.6 dRI—differential refractometer or differential refrac-
Mass concentration detector linearity is determined over the
tive index
mass concentration range of interest according to manufacturer
3.2.7 DLS—dynamic light scattering
instructions on a periodic basis to ensure meaningful recovery
3.2.8 DPBS—Dulbecco’s phosphate buffered saline
determination. This test method presumes that primary instru-
ment qualification has been performed by the manufacturer or
3.2.9 FF—focus flow
according to their recommendations.
3.2.10 FWHM—full width at half maximum
3.2.11 IF—injection flow; sample injection 5. Significance and Use
3.2.12 MALS—multi-angle (static) light scattering
5.1 Liposomal formulations for the treatment of cancer and
other diseases are the most common form of nanotechnology-
3.2.13 MD—multi-detector
enabled drug products submitted for market approval and in
3.2.14 MWCO—molecular weight cut-off
clinical application at the present time. The accurate charac-
3.2.15 PBS—phosphate buffered saline
terization of their physical-chemical properties is critical to
3.2.16 PES—polyethersulfone
support the development and assessment of such products (2).
In particular, size, size distribution, shape, and physical stabil-
3.2.17 PSL—polystyrene latex (sphere, bead)
ity are key properties (among others) that have been widely
3.2.18 RC—regenerated cellulose
identified as critical quality attributes (CQAs) for liposomal
3.2.19 R—retention ratio
drug products.
3.2.20 R —radius of gyration (root mean square radius) 5.1.1 Asymmetrical-flow field-flow fractionation (AF4) is a
g
chromatographic-like technique that uses hydrodynamic forces
3.2.21 R —sphere-equivalent hydrodynamic radius
h
to gently separate analytes into their component populations
3.2.22 R(%)—analyte recovery
according to size and diffusivity (3 and 4). The fractionated
3.2.23 S—selectivity
sample then passes through one or more online detectors
3.2.24 t —void time chosen specifically for the application requirements. The com-
bination of low-shear separation, tolerance for complex
3.2.25 t —retention time
R
matrices, and exceptionally broad size range make AF4 a
3.2.26 UV-Vis—ultraviolet-visible (wavelength range)
technique of choice for application to nanotechnology-enabled
3.2.27 w—channel thickness (nominal as defined by a re-
drug products such as liposomes (13, 5-9).
movable spacer or as defined by a fixed-height channel with no
5.1.2 Multi-detector AF4 is suitable for research and
spacer)
development, manufacturing quality control, product stability/
3.2.28 XF—cross flow shelf-life testing and regulatory assessments.
5.1.3 There are multiple assumptions inherent in the appli-
4. Summary of Test Method
cation of MD-AF4, including the appropriateness of models
4.1 Liposomal drug products are diluted into filtered PBS to used to interpret online light scattering data and the compat-
achieve a total lipid concentration of 1 mg ⁄mL. The diluted test ibility of the analyte and matrix with the membrane that forms
specimen is injected into the MD-AF4 system using PBS as the the surface of the accumulation wall in the fractionation
mobile phase. A total injected lipid mass of 25 μg to 50 μg is channel. Other assumptions are detector or analyte specific.
targeted.
5.2 Chemical components of the mobile phase must not
4.2 Test specimens are analyzed with and without applied induce agglomeration of liposomes or otherwise significantly
cross flow and focusing in order to estimate analyte recovery. alter their physical properties.
4.3 Particle size (root mean square radius and, optionally, 5.3 Discretion should be used in the interpretation of size
hydrodynamic radius) is measured online during elution under data obtained by different scattering detectors using different
cross flow following a standardized procedure (1) that is scattering models and modes of analysis.
generally compliant with ISO/TS 21362.
5.4 The Current Good Manufacturing Practice for Finished
4.4 Prior to sample analysis, instrument system perfor- Pharmaceuticals (see 21 CFR 211.194(a)(2)) and the ICH
mance is verified using BSA in PBS mobile phase. Traceable Harmonized Tripartite Guideline on Validation of Analytical
PSL spheres (for the applicable size range) can be used with an Procedures Q2(R1) (10), state that the suitability of all test
appropriate mobile phase (see Appendix X3) to verify that the methods shall be verified under actual conditions of use.
E3409 − 24
5.5 MD-AF4 can be compared with batch mode dynamic 6.6 This test method is designed to maintain physiological
light scattering (DLS) for the determination of liposome mean pH and osmolarity during analysis using PBS as the principal
size and size distribution, where MD-AF4 provides deconvo- medium. Differences in the osmotic concentration or tonicity
lution of complex mixtures yielding a more accurate assess- of the liposome drug product and the medium or mobile phase
into which the product is diluted can potentially cause osmotic
ment of the populations present. Batch mode DLS (see, for
instance, Test Method E3247) provides a rapid, low-cost stress-induced changes to liposome size (shrinkage or swell-
approach that can be used to screen materials prior to analysis ing) and other structural transformations. Such effects are more
by MD-AF4. The latter requires substantially more analyst likely to occur in low electrolyte media and increase in
time and effort along with appropriate training and expertise. likelihood with increasing liposome size. If significant struc-
Qualitatively, MD-AF4 offers greater insight into the physical tural changes due to osmotic stress are suspected, the analyst
complexity of a sample via the fractionation process in may wish to consider a different methodology or evaluate
combination with multiple detectors. modifications of the mobile phase that allow osmotic matching
(Note 1). Any significant deviations from the proscribed
6. Interferences method must be fit for purpose and clearly stated in the analysis
report.
6.1 Most common sources of interference that can adversely
NOTE 1—To test for the presence of significant osmotic stress-induced
impact batch measurements of liposomes using light
physical changes, the analyst can evaluate the test material using, for
scattering-based methods are mitigated by the fractionation
example, a serial dilution of PBS.
process in AF4, which yields data from nearly monodisperse
population intervals.
7. Apparatus
6.2 Sample matrix components (for example, tonicity 7.1 See Annex A1 for a detailed description of the MD-AF4
agents, surfactants or other molecular species) can interfere instrumentation required for this test method. See Appendix
with the analysis of analyte size if not separated from the X4 for schematic drawings representing three basic channel/
analyte with sufficient resolution. This is generally not an issue flow designs in current or potential commercial use.
for liposomes due to their larger size compared with common
7.2 AF4 Trapezoidal Channel with a nominal channel thick-
sample matrix components.
ness of 350 μm (either fixed-height or using a replaceable
plastic spacer) and a nominal channel length between approxi-
6.3 Sample matrix components can impact the calculated
mately 140 mm and 300 mm.
recovery, as sufficiently small species can pass through the
7.2.1 If a fixed-height channel with a different nominal
accumulation wall membrane during focusing and field-on
height is used, it is the user’s responsibility to verify it is fit for
elution. This loss could reduce the mass concentration detector
purpose and to report this change.
signal and thereby reduce the apparent recovery. This effect is
significant only when the mass concentration detector is
7.3 Mechanical Pipettes and Disposable Pipette Tips, rang-
sensitive to these species (that is, they absorb strongly at
ing from 2 μL to 10 mL.
280 nm in the UV detector). This potential interference may be
7.4 Solvent Reservoir Bottles, 1 L or larger.
evaluated offline using centrifugal filtration or dialysis to
7.5 LC-MS Grade Autosampler Glass Vials (2 mL) with
separate the liposomal analyte from the interfering matrix
polytetrafluoroethylene silicone septa-fitted caps (if using an
components. The filtrate or dialysate can then be tested for
autosampler in conjunction with the MD-AF4 system—these
absorbance at the operational UV wavelength. When these
are unnecessary if a manual sample injector is used).
components are instead retained by the membrane, it may not
be possible to differentiate their contribution to recovery
7.6 Calibrated pH Meter with pH electrode appropriate for
determination.
aqueous solutions and suspensions.
6.4 A common interference for the application of this test
8. Reagents and Materials
method is analyte-membrane interaction in the fractionation
8.1 Unless otherwise stated, references to water shall be
channel. Such interactions can lead to unacceptably low
understood to mean reagent grade (ultrapure) water (≥
recovery and biased results.
18.2 MΩ cm) conforming to Type I of Specification D1193 and
6.4.1 Memory effects can interfere with subsequent analyses
passed through a ≤ 0.22 μm pore size filter. Commercially
when material is carried over from previous elution runs due
available LC-MS grade water meets this specification for the
principally to adherence on the accumulation wall membrane.
purpose of this test method.
Other surfaces in the fractionation channel, though less likely,
can potentially contribute to this effect. 8.2 Reagent-grade chemicals shall be used in all tests.
Unless otherwise indicated, it is required that all reagents
6.5 Scattering at the UV wavelength used to measure
analyte mass can potentially interfere with the accurate deter-
mination of analyte recovery. Therefore, confirmation of the
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
Standard-Grade Reference Materials, American Chemical Society, Washington,
linearity of UV absorbance at 280 nm over the mass range of
DC. For suggestions on the testing of reagents not listed by the American Chemical
interest is recommended. Confirmation that scattering is not a
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
significant interference can also be accomplished using a
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
second independent mass concentration detector such as dRI. copeial Convention, Inc. (USPC), Rockville, MD.
E3409 − 24
conform to the specifications of the Committee on Analytical uniformly spherical, monomodal and with a very narrow size
Reagents of the American Chemical Society, where such distribution (nearly monodisperse with polydispersity index
specifications are available. Other grades may be substituted, if less than 0.1).
necessary, provided it is first ascertained that the reagent is of
8.5 Hydrophilic polyethersulfone (PES) syringe filters with
sufficiently high purity to be fit for purpose.
0.2 μm or 0.1 μm pore diameter and sterile plastic syringes are
recommended to filter water or mobile phase prior to use. PES
8.3 Mobile Phase—The primary mobile phase or eluent for
filter units (150 mL or larger) with a vacuum connector can be
this test method is phosphate buffered saline (PBS), which
used to conveniently and quickly filter larger volumes of water,
nominally contains 8 g ⁄L sodium chloride (NaCl), 0.2 g ⁄L
buffer or mobile phase. A different filter material can be
potassium chloride (KCl), 1.15 g ⁄L sodium phosphate
substituted if verified to be fit for use.
(Na HPO ) and 0.2 g ⁄L potassium phosphate monobasic
2 4
(KH PO ), with pH nominally 7.2 to 7.4. PBS was chosen
2 4
8.6 Precut trapezoidal 10 kDa MWCO regenerated cellulose
because it provides physiological pH buffering and isotonic
(RC) semipermeable membranes for use in the fractionation
dilution of liposome drug formulations. All mobile phase
channel. Precut membranes are obtained from the instrument
solutions shall be filtered at ≤ 0.22 μm pore size prior to use.
manufacturer to fit their standardized channels.
8.3.1 Commercially available sterile PBS buffer formula-
8.7 Plastic spacer (350 μm nominal thickness) compatible
tions (without calcium, magnesium, glucose, pyruvate, and
with the specific AF4 channel that is used to implement this test
phenol red) can vary in their specific chemical composition, pH
method (for example, polyethylene terephthalate “Mylar” or
and designation (for example, Dulbecco’s PBS or DPBS). The
other manufacturer recommended material is acceptable). If a
test method described here was evaluated using four commer-
fixed-height channel of the same nominal thickness (height) is
cial PBS products with similar compositions and pH values
used, a spacer is not needed.
ranging from 7.2 to 7.4. Results indicate that all tested PBS
products yield statistically identical results for liposome for- 8.8 In addition to those listed above, additional materials or
chemicals might be required by the instrument manufacturer
mulations based on sample recovery, separation efficiency and
measured size (1). We conclude that the specific PBS product for the proper operation, maintenance and primary MALS
formulation used is not critical to the outcome of this test calibration of specific instrument platforms. Follow manufac-
turer instructions where appropriate.
method. Any reagent grade, sterile PBS formulation without
calcium, magnesium, glucose, pyruvate and phenol red, and
with a pH from 7.2 to 7.4, is considered acceptable and fit for 9. Hazards
purpose.
9.1 Appropriate protective measures and disposal proce-
8.3.2 Sterile isotonic saline (sodium chloride, 154 mmol ⁄L
dures are required while handling liposome drug formulations
or 9 g ⁄L—commonly labelled as 0.9 % w ⁄v) was evaluated as
that contain hazardous or toxic components (for example,
a secondary buffer and can be used in place of PBS in this test
encapsulated chemotherapeutic drugs); refer to product safety
method and will yield similar results. The user should note that
information where available.
the pH of saline is slightly acidic (pH 6) and is not buffered.
10. Mobile Phase Preparation
8.4 Quality Controls to Evaluate Instrument Performance:
8.4.1 Bovine serum albumin (BSA) lyophilized powder
10.1 Rinse the solvent reservoir bottles at least three (3)
(≥ 99 % purity, Fraction V) or ampouled BSA concentrate. times with sterile ultrapure water filtered using ≤ 0.22 μm pore
BSA is utilized as a Rayleigh scatterer for normalization of size PES filter. The reservoir bottles should be clean and should
MALS sensors (relative to 90° scattering angle primary cali- be used exclusively for media containing water and simple
bration). BSA is also used as a routine verification material for salts.
overall instrument performance (flow rates, pressure, mem-
10.2 Use commercially available (or in-lab prepared), ster-
brane condition, detector operation, etc.) and to detect changes
ile PBS or (optionally) isotonic saline for the mobile phase.
in performance over time. Refer to Appendix X2 for a general
Dilute commercial concentrates (for example, 10x PBS) using
procedure to prepare BSA stock suspension.
ultrapure filtered water. For preparation of in-lab mobile phase,
8.4.2 Commercially available spherical polystyrene latex
refer to Appendix X1 for details.
(PSL) size standards, with nominal diameters of, for example,
10.2.1 Measure and record the pH of the mobile phase.
60 nm, 125 nm, 200 nm, and 350 nm, can be used to verify
10.3 Filter the mobile phase using ≤ 0.22 μm pore size PES
instrument performance with respect to size measurements
filter. Then rinse the solvent reservoir bottles three (3) times
(MALS and DLS) and selectivity (fractionation). Depending
with filtered mobile phase, before filling to nominal capacity.
on the size of the liposome analyte, other nominal size PSLs
can be chosen to bracket the analyte size. Only the PSL
10.4 Continuous circulation of mobile phase through an
monomer peak is of interest in this context; dimers or other
in-line filter (typical with commercial isocratic pumps) main-
oligomers will elute later in time and can be ignored. Refer to
tains sterile conditions throughout the system. Chemical
Appendix X3 for general procedures to prepare PSL samples
disinfectants, such as sodium azide, are not recommended and
for analysis.
have not been evaluated with the test method described here.
8.4.2.1 Other (non-polystyrene) polymer latices may be Their inclusion requires verification by the user. When instru-
used if fit for purpose. Principally, these particles should be mentation is not being used for analysis, the user should follow
E3409 − 24
manufacturer recommendations for maintaining sterile condi- turer instructions for this procedure and for sealing the channel
tions in the AF4 system, including use of anti-microbial agents. afterwards. If using a fixed-height channel, a spacer is not
required. A larger channel height is not recommended for
11. Sampling and Test Specimens
implementation of this test method; use of a larger channel
11.1 Liposome drug formulations should be stored under
height must be validated by the user as fit for purpose, and will
conditions specified by the manufacturer until needed.
most likely require other changes (for example, flow rates).
Typically, this involves refrigeration at 2 °C to 8 °C.
12.1.5 Install the solvent reservoir containing mobile phase,
and prime the pump(s), including tubing connections and
11.2 Prior to measurement prepare a stock suspension by
detectors according to the instructions provided by the manu-
diluting the native liposome formulation into the mobile phase
facturer.
(PBS) to obtain a final lipid concentration of 1 mg ⁄mL. Store
the stock suspension refrigerated at 2 °C to 8 °C until needed
12.2 Preparation of Online Detectors:
for analysis. Diluted suspensions must be analyzed within 24 h.
12.2.1 Follow manufacturer recommendations for powering
11.2.1 Stock should be prepared in a clean sterile glass or
up the UV-Vis detector prior to the first analysis run. Set the
plastic tube/vial with a cap or seal.
detector wavelength to 280 nm.
11.2.2 For most situations, less than 2 mL of stock suspen-
NOTE 2—This wavelength is generally appropriate for lipid mass
sion will be needed for several MD-AF4 analyses.
detection. If the detector has the capacity to monitor more than one
wavelength or to measure a spectral range in real time (for example, using
11.3 Test specimens are withdrawn from the stock suspen-
a DAD), the user may consider selecting a second wavelength that is
sion using a clean appropriate size injection syringe (for
sensitive to a specific component in order to monitor the concentration of
manual injectors) or using a pipettor with a clean sterile pipette
that component separately from the lipid content (liposome concentra-
tip to transfer test specimen into appropriate LC vials (if using
tion).
an autosampler).
12.2.2 Power up the MALS detector including laser source
11.4 If the purpose of the test is to evaluate stability of the
at least 1 h prior to analysis. Observe average noise level to
liposomes, for example, in physiological media, dilute the
monitor cleanliness of the system. Follow manufacturer rec-
native formulation into the physiological medium to obtain a
ommendations for background noise levels and to address
lipid concentration of 1 mg ⁄mL. The composition of the
unacceptably high noise.
medium must be compatible with analysis by AF4; it is the
12.2.3 If not integrated with the MALS detector, power up
user’s responsibility to verify compatibility.
the DLS detector at least 1 h prior to analysis.
11.4.1 If desired, incubate this stock for a suitable time
12.2.4 Use of other detectors is not prohibited but is beyond
period under relevant conditions. Then follow instructions
the scope of this test method. Follow instrument manufacturer
above to remove or transfer test specimen prior to analysis.
instructions for set-up, operation and performance verification
11.4.2 The incubated test specimen is then fractionated
of unspecified detectors. Alternative mass concentration detec-
using PBS mobile phase in the same manner as native samples
tors must be demonstrated as fit for purpose.
diluted directly into the mobile phase.
12.3 System Operational Verification:
12. Preparation of Apparatus
12.3.1 The operational particle size range for both DLS and
MALS has angle- and flow rate-dependent limitations, and the
12.1 Initial Assembly and System Preparation:
accuracy of MALS-derived size values is dependent on correct
12.1.1 The MD-AF4 system shall be assembled and main-
calibration of the detector. Since DLS and MALS detector
tained according to manufacturer recommendations. Between
performance is integral to this test method, operational perfor-
analyses the system can pump mobile phase at a minimum flow
mance of the complete measurement system must be verified
rate of 0.1 mL ⁄min in a continuous closed loop to maintain
for the size range of interest and using the same or similar
sterility, conserve mobile phase and reduce waste (refer to
fractionation conditions to be used for the liposome test
manufacturer recommendations). Follow manufacturer recom-
specimen (Note 3). This verification need only be performed
mendations to power down for long-term storage when not in
following a significant change to the system configuration,
daily use, and to power up and recondition the system prior to
such as calibration or cleaning of the MALS detector or a
use, and for short-term between-experiment settings.
change to the scattering angle used for the DLS detector, or
12.1.2 This test method presumes that the MD-AF4
when the liposome analyte size range changes substantially.
instrument, including all detectors, the channel and tubing, are
clean and fully operational according to manufacturer specifi-
NOTE 3—It is implicitly understood that the mobile phase used for
cations. Furthermore, all components in contact with liquid verification will likely differ from the mobile phase used for liposome
analysis, requiring a mobile phase exchange and flush following valida-
have been conditioned according to manufacturer instructions
tion.
for use with an aqueous mobile phase.
12.1.3 This test method presumes that the MD-AF4 12.3.2 Verification procedures can vary depending on the
instrument, including detectors, is properly calibrated as re- instrument platform and the objective of the procedure. When
quired and according to manufacturer instructions and specifi- in doubt, follow the manufacturer recommendations. For
cations. verifying the operable size range, verification can be performed
12.1.4 To prepare for a new set of experiments, insert the using PSL spheres (see 8.4.2). PSL is the recommended choice
appropriate size 10 kDa RC membrane into a clean channel for liposome applications because of the spherical shape,
and ensure that a 350 μm spacer is in place. Follow manufac- organic composition, material density, and availability over a
E3409 − 24
wide range of sizes. PSL sizes should be chosen to roughly mended by the instrument manufacturer. In this way, the results
bracket the size range of the analyte where possible (Note 4). can be recorded and compared to previous runs on the same
See Appendix X3 for preparation of PSL stock suspensions. system to detect and document changes in system performance.
To be clear, this step provides system performance verification,
NOTE 4—For routine verification of instrument operation, a single PSL
but does not validate methodology as fit for purpose (with
is sufficient (in which case a size should be chosen that is similar to the
respect to analysis of liposomal formulations).
analyte—125 nm diameter PSL is a good overall choice for many
liposome formulations).
13. Calibration and Standardization
12.3.2.1 Inject an appropriate mass of PSL that will provide
13.1 To ensure SI-traceable results, relevant instrument,
sufficient signal while avoiding detector saturation; typically,
detector or test parameters must be measured with traceably
5 μg to 50 μg is sufficient depending on PSL size (Note 5).
calibrated tools (for example, cell temperature, known laser
Determine the mean R and R at FWHM of the main peak (as
g h
wavelength and scattering angle). This is typically performed
traced by the 90° scattering intensity signal) (Note 6). Ignore
by the manufacturer during production, prior to delivery or
peaks associated with dimers or larger aggregates. Determine
periodically as part of a maintenance program.
t and selectivity (if more than one size PSL is used). Compare
R
to previous experiments if data are available and record results
13.2 The user can ensure accuracy by following the instru-
for future reference. As a rule of thumb, the measured mean
ment manufacturer instructions for calibration of the MALS
values for (2×R ) or (2×=5⁄3R ) should be within the stated detector and by periodic verification of instrument performance
h g
uncertainty range for the sphere-equivalent mean diameter of using, for example, traceable or certified PSL spheres under
the PSL. controlled conditions and following a standard operating pro-
NOTE 5—Scattering intensity increases exponentially with PSL
cedure as defined by the user or manufacturer for the specific
diameter, such that the necessary injected mass will decrease with
instrument configuration used.
increasing size.
13.2.1 Primary calibration and normalization of the MALS
NOTE 6—For verification purposes, a sphere model can be used if
detector shall be performed periodically, as recommended by
available (yields the geometric diameter or radius). If a sphere model is
the instrument manufacturer and following the manufacturer
not available, the Berry model can be used to calculate R , which can then
g
recommended procedure.
be converted to the equivalent sphere using R5=5⁄3R .
g
13.2.2 The DLS detector does not require calibration, but
12.3.3 Prior to starting a series of measurements or after
performance should be verified simultaneously with the MALS
replacement of the channel membrane or following any sig-
detector using PSL spheres (or other appropriate material as
nificant maintenance on the AF4 system, inject 50 μg to 150 μg
recommended by the manufacturer) of an appropriate size or
of BSA in PBS mobile phase (refer to Appendix X2 for
size range.
preparation of BSA stock solution and recommended analysis
NOTE 8—Note that medium viscosity is an input parameter for the
parameters) as a quality control material to verify system
calculation of R from DLS. The analyst should ensure that the correct
h
performance under the user’s or manufacturer’s predefined
temperature dependent viscosity is selected for DLS measurements. As a
standard operating procedure. Following manufacturer
rule of thumb, the viscosity of PBS is roughly 2 % higher than water at
instructions, the BSA test can also be used to account for
temperatures from 20 °C to 25 °C. Refer to Test Method E3247.
detector delays and correct for band broadening.
14. Conditioning
12.3.3.1 Process the data obtained during the performan
...