ASTM E3042-16(2024)

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: E3042 − 16 (Reapproved 2024)
Standard Practice for
Process Step to Inactivate Rodent Retrovirus with Triton
1,2
X-100 Treatment
This standard is issued under the fixed designation E3042; 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 ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
1.1 This practice assures effective inactivation of ≥4 log of
mendations issued by the World Trade Organization Technical
infectious rodent retrovirus (that is, reduction from 10 000 to 1
Barriers to Trade (TBT) Committee.
infectious rodent retrovirus or removal of 99.99 % of infectious
rodent retroviruses) in the manufacturing processes of mono-
2. Terminology
clonal antibodies or immunoglobulin G (IgG) Fc fusion pro-
teins manufactured in rodent-derived cell lines that do not
2.1 Definitions of Terms Specific to This Standard:
target retroviral antigens. Rodent retrovirus is used as a model
2.1.1 clarified, cell free intermediate, n—in-process pool
for rodent cell substrate endogenous retrovirus-like particles
located downstream of the cell clarification unit operation(s),
potentially present in the production stream of these proteins.
which should include a filtration step of ≤0.2 μm nominal pore
size, and upstream of the initial purification step in the
1.2 The parameters specified for this practice are
clarification, Triton X-100 detergent concentration, hold time, purification process of a monoclonal antibody or IgG Fc fusion
protein.
pH, and inactivation temperature.
2.1.1.1 Discussion—Cell clarification unit operations are
1.3 This practice can be used in conjunction with other
performed on the cell culture supernatant. Cell clarification
clearance or inactivation unit operations that are orthogonal to
unit operations can be one or more of the following opera-
this inactivation mechanism to achieve sufficient total process
tion(s): microfiltration, centrifugation, depth filtration, or
clearance or inactivation of rodent retrovirus.
flocculation, or combination thereof. The primary purpose of
1.4 This detergent inactivation step is performed on a
cell clarification unit operation(s) is to remove cells used to
clarified, cell-free intermediate of the monoclonal antibody or
generate monoclonal antibody or IgG Fc fusion protein and
IgG Fc fusion protein.
some proportion of cellular debris from the cell culture
1.5 The values stated in SI units are to be regarded as
supernatant before the initial purification step. All clarification
standard. No other units of measurement are included in this
steps must include ≤0.2 μm nominal pore size filtration to
standard.
minimize the presence of virus aggregates, prior to detergent
inactivation. Freezing or prolonged storage between ≤0.2 μm
1.6 This standard does not purport to address all of the
filtration and detergent inactivation should be avoided.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
2.1.2 enveloped virus, n—viruses in which the nucleic acid
priate safety, health, and environmental practices and deter-
component of the virus is surrounded by a lipid containing
mine the applicability of regulatory limitations prior to use.
envelope acquired from the host cell during virus assembly and
1.7 This international standard was developed in accor-
budding.
dance with internationally recognized principles on standard-
2.1.2.1 Discussion—Some examples of enveloped viruses
are from the families orthomyxoviridae (influenza), paramyxo-
This practice is under the jurisdiction of ASTM Committee E55 on Manufac-
viridae [mumps and measles], retroviridae [human immuno-
ture of Pharmaceutical and Biopharmaceutical Products and is the direct responsi-
deficiency virus (HIV) and murine leukemia virus (MuLV)],
bility of Subcommittee E55.12 on Process Applications.
and herpesviridae [human herpes virus (HHV), varicella-zoster
Current edition approved Feb. 1, 2024. Published February 2024. Originally
approved in 2016. Last previous edition approved in 2016 as E3042 – 16. DOI:
virus (VZV), and pseudorabies virus (PRV)].
10.1520/E3042-16R24.
2.1.3 hold time, n—amount of time, after sufficient mixing
Triton X-100 is a trademark of The Dow Chemical Company, Midlands,
Michigan, http://www.dow.com. The sole source of manufacture of the material
takes place, that the biological drug intermediate and retrovirus
known to the committee at this time is The Dow Chemical Company. If you are
interact with a specific chemical, in this case, the amount of
aware of alternative suppliers, please provide this information to ASTM Interna-
time the biological drug intermediate and retrovirus interact
tional Headquarters. Your comments will receive careful consideration at a meeting
of the responsible technical committee, which you may attend. with the Triton X-100.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3042 − 16 (2024)
2.1.3.1 Discussion—Demonstration of sufficient mixing is 2.1.11 inactivation temperature, n—temperature (°C) of ma-
the responsibility of the manufacturer. trix in the container holding the Triton X-100 and the clarified,
cell-free intermediate.
2.1.4 immunoglobulin G, IgG, n—antibody molecule com-
2.1.12 retrovirus, n—ribonucleic acid (RNA) virus that is
posed of four peptide chains—two gamma heavy chains and
propagated in a host cell using the reverse transcriptase enzyme
two light chains.
to produce deoxyribonucleic acid (DNA) from its RNA ge-
2.1.4.1 Discussion—Each IgG has two antigen binding
nome.
sites. IgG constitutes 75 % of serum immunoglobulins in
2.1.12.1 Discussion—The DNA is then incorporated into the
humans. IgG molecules are synthesized and secreted by plasma
host’s genome by an integrase enzyme. The virus thereafter
B cells. There are four IgG subclasses (IgG1, 2, 3, and 4) in
replicates as part of the host cell’s DNA. Retroviruses are
humans named in order of their abundance in serum (IgG1
enveloped viruses that belong to the viral family Retroviridae.
being the most abundant).
2.1.13 Triton X-100 (polyethylene glycol p-(1,1,3,3-
2.1.5 immunoglobulin G (IgG) fusion protein, n—dimeric
tetramethylbutyl)-phenyl ether), n—non-ionic surfactant; a liq-
proteins comprised of two monomers, each monomer consist-
uid at room temperature.
ing of a peptide sequence (usually a human receptor-like
2.1.13.1 Discussion—Triton X-100 is also known as poly-
protein or protein fragment) fused to a human IgG antibody Fc
ethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, oc-
domain.
tyl phenol ethoxylate, Octylphenol Ethoxylate (non-ionic), and
2.1.6 effective viral clearance, n—a viral clearance unit
Octoxynol-9. The CAS number for Triton X-100 is 9002-93-1.
operation that removes or inactivates ≥4 log reduction value
In this practice, the chemical polyethylene glycol p-(1,1,3,3-
of virus.
tetramethylbutyl)-phenyl ether, CAS number 9002-93-1, will
be referred to as Triton X-100.
2.1.6.1 Discussion—Inactivation requires a loss of infectiv-
ity.
2.1.14 Triton X-100 concentration, n—percentage of Triton
X-100 (% weight : volume) in the Triton X-100 detergent
2.1.7 log reduction value, LRV, n—log reduction is
10 10
solution.
typically used to describe the degree of reduction of an
organism population, in this case, rodent retrovirus, or other
3. Significance and Use
enveloped virus, by the treatment process.
3.1 Rodent-derived cell lines are widely used in the produc-
2.1.7.1 Discussion—Each log reduction represents a 90 %
tion of biopharmaceutical drugs such as mAbs and Fc fusion
reduction in the organism population so a process shown to
proteins. These cell lines have been shown to contain genes
achieve a “6 log reduction” will reduce a population from a
encoding endogenous retroviral-like particles or endogenous
million organisms to one.
retrovirus. Despite the lack of evidence for an association
2.1.8 modular viral validation, n—modular clearance study
between such rodent retroviruses and disease in humans, the
is one that demonstrates virus removal or inactivation by
potential contamination of human therapeutics raises safety
individual unit operations during the purification process
concerns for biopharmaceutical drugs. Additionally, adventi-
(column chromatography, filtration, pasteurization, solvent/
tious agents such as viruses can be introduced into a biophar-
detergent, low pH, and so forth).
maceutical drug substance manufacturing process from other
2.1.8.1 Discussion—Each unit operation, or module, in the sources, and potential safety issues can be attributed to these
purification scheme may be studied independently of the other
potential unknowns. For these reasons, effective viral clearance
modules. Different model monoclonal antibodies (mAbs) may is an essential aspect of an integrated approach combining
be used to demonstrate viral clearance in different modules, if
safety testing and process characterization which ensures virus
necessary. If the purification process parameters used in the safety for biopharmaceutical drug products made using rodent
manufacturing of a mAb product differs at any of the virus
cell lines.
removal or inactivation modules from the model mAb, this
3.2 Solvent/detergent inactivation has been widely used for
module shall be studied independently from the model. The
decades to inactivate enveloped viruses in blood plasma
other, identical modules in the procedure may be extrapolated
derived biopharmaceutical therapies (1-3). Solvent/detergent
to the product mAb.
systems using the detergents Triton X-100 or Polysorbate 80
2.1.9 monoclonal antibody, mAb, n—monospecific, recom- along with the organic solvent tri(n-butyl)phosphate (TNBP)
binant antibody manufactured using a productio
...