ASTM E3259-22

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: E3259 − 22
Standard Practice for
Process to Remove Retroviruses by Small Virus Retentive
Filters
This standard is issued under the fixed designation E3259; 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 mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.1 This practice assures 6.0 log removal of retrovirus (for
example, MuLV).
2. Terminology
1.2 This practice is applicable to monoclonal antibody
2.1 Definitions of Terms Specific to This Standard:
(mAb), immunoglobulin G (IgG) fusion proteins, recombinant
2.1.1 bacteriophage PP7, n—RNA bacteriophage that in-
proteins, or other proteins produced using mammalian cell
fects Pseudomonas aeruginosa bacteria that has a size of
lines (for example, Chinese hamster ovary (CHO), murine
approximately 30 nm to 33 nm, often used as a surrogate for
hybridomas, murine myelomas, or human embryonic kidney
parvovirus in virus retentive filter studies, but not for regula-
(HEK) 293).
tory clearance claims.
1.3 The step is performed on cell-free intermediates.
2.1.2 bacteriophage PR772, n—double-stranded DNA bac-
1.4 The log removal claim for retrovirus by small virus
teriophage that infects Escerichia coli bacteria that has a size of
retentive filters can be used in conjunction with other clearance
approximately 80 nm, often used as a surrogate for retrovirus
unit operations (for example, low pH inactivation, or inactiva-
in virus retentive filter studies, but not for regulatory clearance
tion of virus by surfactant) to assure sufficient total process
claims.
clearance of potential virus contaminants, which would be
supportive of early phase (clinical phase 1 or phase 2a trials) 2.1.3 log reduction value (LRV) or log reduction factor
regulatory filings.
(LRF), n—used to describe the degree of reduction of a
population; for virus filtration, LRV/LRF is calculated by
1.5 Retrovirus removal claim by filtration is limited to small
comparing the virus quantity before and after filtration.
virus retentive filters, as defined in the PDA Technical Report
–1
2.1.3.1 Discussion—Each log reduction (10 ) represents a
Virus Filtration (1) in the context of this standard.
90 % reduction in the population. For example, a process
1.6 The values stated in SI units are to be regarded as
–6
shown to achieve a 6-log reduction (10 ) will reduce a
standard. No other units of measurement are included in this
population from a million (10 ) to 1. The calculation for log
standard.
reduction is [log of total virus quantity in feed material or
1.7 This standard does not purport to address all of the
load] minus [log total virus quantity in filtrate or filtered
safety concerns, if any, associated with its use. It is the
product].
responsibility of the user of this standard to establish appro-
2.1.4 modular virus clearance validation, n—a modular
priate safety, health, and environmental practices and deter-
viral clearance study is one that demonstrates virus removal
mine the applicability of regulatory limitations prior to use.
and/or inactivation at individual steps during the purification
1.8 This international standard was developed in accor-
process (column chromatography, filtration, heat treatment,
dance with internationally recognized principles on standard-
solvent/detergent treatment, low pH treatment, and so forth).
ization established in the Decision on Principles for the
2.1.4.1 Discussion—Each module, or unit operation, in the
Development of International Standards, Guides and Recom-
purification scheme may be studied independently of the other
modules. Different model proteins may be used to demonstrate
viral clearance in different modules, if necessary. If the
This practice is under the jurisdiction of ASTM Committee E55 on Manufac-
purification process of a specific product (protein) differs at any
ture of Pharmaceutical and Biopharmaceutical Products and is the direct responsi-
bility of Subcommittee E55.12 on Process Applications.
Current edition approved Nov. 1, 2022. Published November 2022. DOI:
10.1520/E3259-22.
The boldface numbers in parentheses refer to a list of references at the end of
this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3259 − 22
of the virus removal or inactivation modules from the model for virus clearance when well designed. Size exclusion has
protein, this module must be studied independently from the been shown to be the primary mechanism of virus removal by
model. (1) virus retentive filtration, that is, larger viruses are more easily
retained than smaller viruses such as parvoviruses (4, 5). Large
2.1.5 monoclonal antibody, (mAb), n—antibodies that have
virus retention has also been shown to be insensitive to process
affinity for specific antigen(s) and are made from a master cell
fluid characteristics such as protein type, protein concentration,
bank or working cell bank from a cloned from a parent cell.
pH, and ionic strength (4, 6, 7, 8, 9, 10). In contrast, for small
2.1.6 parvovirus, n—small non-enveloped, single-stranded
viruses, aspects like flow pausing and/or flux decay can impact
DNA virus, approximately 18 nm to 26 nm in diameter (2).
clearance (4, 6, 11).
2.1.6.1 Discussion—Minute virus of mice (MMV) and por-
3.3 Large virus retentive filters, or retrovirus filters, are
cine parvovirus (PPV) are small non-enveloped parvoviruses
tested for removal of larger enveloped viruses like retrovirus or
(approximately 18 nm to 26 nm) (3, 4). MMV and PPV are
MuLV (80 nm to 100 nm) and have undetectable levels of the
used in viral clearance studies as a model virus for small
large bacteriophage PR772 (64 nm to 82 nm) (1). Small virus
parvoviruses. Due to the mechanism of virus retention on virus
retentive filters, or parvovirus filters, are designed to remove
retentive filters, the size of parvoviruses is regarded as a
parvovirus, like MMV (18 nm to 26 nm) (1). Since size
worst-case challenge for small virus retention filters (4).
exclusion has been demonstrated as the mechanism of virus
2.1.7 recombinant protein, n—protein produced using re-
retention, retroviruses, which are three to four times larger than
combinant DNA technology, expressed in living cells.
parvoviruses, should be large enough to be completely
2.1.8 retrovirus, n—enveloped, single-stranded RNA virus
retained, with undetectable levels of retrovirus in the filtrate, by
that is propagated in host cell using a reverse transcriptase
all small virus retentive filters designed to remove parvovirus.
enzyme to produce viral cDNA from its RNA genome. Viral
3.4 Numerous published studies and reviews encompassing
cDNA is incorporated into the host genome by viral integrase
data from the last 20 years have shown both large and small
enzyme. Once the viral genome is integrated, virus can
virus retentive filters are effective and consistent for removal of
thereafter be replicated as part of the host cell DNA (2).
retrovirus. In published reviews of regulatory submissions
2.1.8.1 Discussion—Murine leukemia virus (MuLV) is a
from 1990 through 2010, rare occurrences of retrovirus break-
large, enveloped virus (approximately 80 nm to 110 nm) be-
through did occur across both large and small virus retentive
longing to the retroviridae family (3, 4). MuLV, and various
filters. These anomalies, however, were not resolved and could
subtypes of MuLV, including xenotropic (xMuLV), ecotropic
be attributed to study design, experimental artifacts, or limita-
(eMuLV), and amphotropic (aMuLV), are used in viral clear-
tions of the meta-analyses performed on the regulatory sub-
ance studies to model endogenous retrovirus or retrovirus-like
mission (12). In a summary of 89 submissions to Paul Ehrlich
particles.
Institute (PEI), processes using either large or small virus
2.1.9 small virus retentive filter, n—also known as parvovi-
retentive filters showed no detection of any infectious particles
rus retentive filters and PP7-LRF4 filters, filters that are
from large viruses (12). A collection of viral filtration results
designed and claimed by the manufacturer or validated substi-
across eight biopharmaceutical companies showed no large
tute vendor through filter lot release testing to retain parvovirus
virus breakthrough across any small virus retentive filter for all
(18 nm to 26 nm) and be capable of retention of >4 log of
198 experiments reviewed (7). Additionally, a recent review of
bacteriophage PP7 (30 nm to 33 nm) across three lots of virus
20 plus years of small virus retentive filter experiments from
filters at load ratio of at least 50 L ⁄m and meet all of the
two viral clearance testing houses showed only 0.61 % (14 out
stipulations defined in the PDA Technical Report Virus Filtra-
of 2311 experiments) viral filtration studies performed with
tion (1).
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