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ASTM E2888-12

(Practice)

Standard Practice for Process for Inactivation of Rodent Retrovirus by pH

Standard Practice for Process for Inactivation of Rodent Retrovirus by pH

SCOPE
1.1 This practice assures 5 log10 inactivation of non-defective C-type retroviruses, which are endogenous to murine hybridoma and CHO cells and are potentially present in the production stream of biopharmaceutical processes that use rodent derived cell culture.  
1.2 The process parameters specified in this practice consistently assure 5 log10 inactivation of murine retrovirus by adjusting the pH of a process solution after initial affinity capture chromatography purification.  
1.3 This practice is applicable to mAb, IgG fusion, or other recombinant proteins produced from rodent cell lines (for example, CHO or murine hybridoma), which do not target retroviral proteins. Additionally, the low pH step is performed on a cell-free intermediate, post initial capture using protein A chromatography.  
1.4 The 5 log10 inactivation of murine retrovirus claimed by using this practice will be utilized in conjunction with other clearance unit operations (for example, chromatography and virus retentive filtration) to assure sufficient total process clearance of murine retroviruses, which will be supportive of early phase regulatory filings.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

General Information

Status
Historical
Publication Date
31-Jul-2012
ICS
07.100.01 - Microbiology in general
Technical Committee
E55 - Manufacture of Pharmaceutical and Biopharmaceutical Products
Drafting Committee
E55.04 - General Biopharmaceutical Standards
Current Stage
Ref Project

Relations

Referred By
ASTM E3326-22 - Standard Guide for Application of Continuous Manufacturing (BioCM) in the Biopharmaceutical Industry
Effective Date
01-Aug-2012

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ASTM E2888-12 - Standard Practice for Process for Inactivation of Rodent Retrovirus by pHASTM E2888-12 - Standard Practice for Process for Inactivation of Rodent Retrovirus by pH
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ASTM E2888-12 - Standard Practice for Process for Inactivation of Rodent Retrovirus by pH
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E2888 − 12
Standard Practice for
1
Process for Inactivation of Rodent Retrovirus by pH
This standard is issued under the fixed designation E2888; 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 2.1.2 immunoglobulin G (IgG)—an antibody molecule com-
posed of four peptide chains — two γ heavy chains and two
1.1 This practice assures 5 log10 inactivation of non-
light chains.
defective C-type retroviruses, which are endogenous to murine
2.1.2.1 Discussion—Each IgG has two antigen binding
hybridoma and CHO cells and are potentially present in the
sites. IgG constitutes 75 % of serum immunoglobulins in
production stream of biopharmaceutical processes that use
humans.IgGmoleculesaresynthesizedandsecretedbyplasma
rodent derived cell culture.
B cells. There are four IgG subclasses (IgG1, 2, 3, and 4) in
1.2 The process parameters specified in this practice con-
humans, named in order of their abundance in serum (IgG1
sistently assure 5 log10 inactivation of murine retrovirus by
being the most abundant). Only human IgG1, IgG2, and IgG4
adjusting the pH of a process solution after initial affinity
show significant affinity to protein A.
capture chromatography purification.
2.1.3 log10 reduction value (LRV)—typically used to de-
scribe the degree of reduction of a population, in this case
1.3 This practice is applicable to mAb, IgG fusion, or other
rodent retrovirus, by the treatment process.
recombinant proteins produced from rodent cell lines (for
example, CHO or murine hybridoma), which do not target -1
2.1.3.1 Discussion—Each log reduction (10 ) represents a
retroviral proteins. Additionally, the low pH step is performed
90 % reduction in the population. So a process shown to
on a cell-free intermediate, post initial capture using proteinA -6
achieve a 6-log reduction (10 ) will reduce a population from
chromatography. 6
a million (10)to1.
2.1.4 monoclonal antibody (mAb)—monospecificantibodies
1.4 The 5 log10 inactivation of murine retrovirus claimed
which have affinity for the same antigen and are made from a
by using this practice will be utilized in conjunction with other
master cell bank, cloned from a parent cell.
clearance unit operations (for example, chromatography and
virus retentive filtration) to assure sufficient total process
2.1.5 murine leukemia virus (MuLV)—retroviruses named
clearance of murine retroviruses, which will be supportive of
for their ability to cause cancer in murine (mouse) hosts.
early phase regulatory filings.
2.1.5.1 Discussion—MuLV is a member of the genus Gam-
maretrovirus. MuLV is an enveloped spherical RNA virus
1.5 The values stated in SI units are to be regarded as
which has a diameter of 80–110 nm and has low chemical
standard. No other units of measurement are included in this
resistance. MuLV is used as a model for non-defective C-type
standard.
endogenous retrovirus or retrovirus like particles produced by
murine hybridoma and CHO cell lines. MuLV is used to assess
2. Terminology
rodent retrovirus clearance of protein purification processes
2.1 Definitions of Terms Specific to This Standard:
that use rodent cells for production.
2.1.1 IgG fusion protein—a dimeric protein comprised of
2.1.6 recombinant protein—produced from the expression
two monomers, each monomer consisting of a peptide se-
of recombinant DNA within living cells.
quence (usually a human receptor-like protein or protein
2.1.6.1 Discussion—Recombinant DNA is genetically engi-
fragment) fused to the carboxyl-terminal of the Fc-domain of a
neered by inserting foreign DNA into the DNA of an appro-
human IgG antibody.
priate host so that the foreign DNAis replicated along with the
2.1.1.1 Discussion—Dimerization occurs by way of the Fc
host DNA.
domain.
2.1.7 retrovirus—an RNA virus that is propagated in a host
cell using the reverse transcriptase enzyme to produce DNA
from its RNA genome.
1
This practice is under the jurisdiction of ASTM Committee E55 on Manufac-
2.1.7.1 Discussion—DNA is then incorporated into the
ture of Pharmaceutical and Biopharmaceutical Products and is the direct responsi-
host’s genome by an integrase enzyme. The virus is thereafter
bility of Subcommittee E55.04 on General Biopharmaceutical Standards.
replicated as part of the host cell’s DNA. Retroviruses are
Current edition approved Aug. 1, 2012. Published September 2012. DOI:
10.1520/E2888-12. enveloped viruses that belong to the viral family Retroviridae.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

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ASTM E2888-12(2019)(Practice)
Standard Practice for Process for Inactivation of Rodent Retrovirus by pH

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1.1 This practice assures 5 log10 inactivation of non-defective C-type retroviruses, which are endogenous to murine hybridoma and CHO cells and are potentially present in the production stream of biopharmaceutical processes that use rodent derived cell culture.  
1.2 The process parameters specified in this practice consistently assure 5 log10 inactivation of murine retrovirus by adjusting the pH of a process solution after initial affinity capture chromatography purification.  
1.3 This practice is applicable to mAb, IgG fusion, or other recombinant proteins produced from rodent cell lines (for example, CHO or murine hybridoma), which do not target retroviral proteins. Additionally, the low pH step is performed on a cell-free intermediate, post initial capture using protein A chromatography.  
1.4 The 5 log10 inactivation of murine retrovirus claimed by using this practice will be utilized in conjunction with other clearance unit operations (for example, chromatography and virus retentive filtration) to assure sufficient total process clearance of murine retroviruses, which will be supportive of early phase regulatory filings.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 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.

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3.1 Rodent-derived cell lines are widely used in the production of biopharmaceutical drugs such as mAbs and Fc fusion proteins. These cell lines have been shown to contain genes encoding endogenous retroviral-like particles or endogenous retrovirus. Despite the lack of evidence for an association between such rodent retroviruses and disease in humans, the potential contamination of human therapeutics raises safety concerns for biopharmaceutical drugs. Additionally, adventitious agents such as viruses can be introduced into a biopharmaceutical drug substance manufacturing process from other sources, and potential safety issues can be attributed to these potential unknowns. For these reasons, effective viral clearance is an essential aspect of an integrated approach combining safety testing and process characterization which ensures virus safety for biopharmaceutical drug products made using rodent cell lines.  
3.2 Solvent/detergent inactivation has been widely used for decades to inactivate enveloped viruses in blood plasma derived biopharmaceutical therapies (1-3).3 Solvent/detergent systems using the detergents Triton X-100 or Polysorbate 80 along with the organic solvent tri(n-butyl)phosphate (TNBP) have been used to inactivate enveloped viruses by disrupting the viral envelope thereby reducing the ability of the enveloped virus to attach to and then infect the host cell (4 and 5).  
3.3 Most manufacturers of mAbs, recombinant proteins, and Fc fusion proteins have focused on viral inactivation methods using the detergent Triton X-100 or Polysorbate 80 in the absence of TNBP (6), which can interfere with subsequent bioprocessing steps. The ability of the detergents alone to inactivate retroviruses has been demonstrated in monoclonal antibodies produced in rodent-derived cell lines (6-9). At a 2011 workshop devoted to viral clearance steps used in bioprocessing (7), investigators from one firm showed incubation with 0.2 % Triton X-100 for 60 min of hold time at a...
SCOPE
1.1 This practice assures effective inactivation of ≥4 log10 of infectious rodent retrovirus (that is, reduction from 10 000 to 1 infectious rodent retrovirus or removal of 99.99 % of infectious rodent retroviruses) in the manufacturing processes of monoclonal antibodies or immunoglobulin G (IgG) Fc fusion proteins manufactured in rodent-derived cell lines that do not target retroviral antigens. Rodent retrovirus is used as a model for rodent cell substrate endogenous retrovirus-like particles potentially present in the production stream of these proteins.  
1.2 The parameters specified for this practice are clarification, Triton X-100 detergent concentration, hold time, pH, and inactivation temperature.  
1.3 This practice can be used in conjunction with other clearance or inactivation unit operations that are orthogonal to this inactivation mechanism to achieve sufficient total process clearance or inactivation of rodent retrovirus.  
1.4 This detergent inactivation step is performed on a clarified, cell-free intermediate of the monoclonal antibody or IgG Fc fusion protein.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
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 and health practices and determine the applicability of regulatory limitations prior to use.

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1.1 This test method is used for growing a reproducible (1)2 Pseudomonas aeruginosa biofilm in a continuously stirred tank reactor (CSTR) under medium shear conditions. In addition, the test method describes how to sample and analyze biofilm for viable cells.  
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1.1 Microbiological test methods present challenges that are unique relative to chemical or physical parameters, because microbes proliferate, die off and continue to be metabolically active in samples after those samples have been drawn from their source.  
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5.1 Vegetative biofilm bacteria are phenotypically different from suspended planktonic cells of the same genotype. Biofilm growth reactors are engineered to produce biofilms with specific characteristics. Altering either the engineered system or operating conditions will modify those characteristics. The goal in biofilm research and efficacy testing is to choose the growth reactor that generates the most relevant biofilm for the particular study.  
5.2 The purpose of this test method is to direct a user in how to grow, treat, sample and analyze a Pseudomonas aeruginosa biofilm using the MBEC Assay. Microscopically, the biofilm is sheet-like with few architectural details as seen in Harrison et al (6). The MBEC Assay was originally designed as a rapid and reproducible assay for evaluating biofilm susceptibility to antibiotics (2). The engineering design allows for the simultaneous evaluation of multiple test conditions, making it an efficient method for screening multiple disinfectants or multiple concentrations of the same disinfectant. Additional efficiency is added by including the neutralizer controls within the assay device. The small well volume is advantageous for testing expensive disinfectants, or when only small volumes of the disinfectant are available.
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1.1 This test method specifies the operational parameters required to grow and treat a Pseudomonas aeruginosa biofilm in a high throughput screening assay known as the MBEC (trademarked)2 (Minimum Biofilm Eradication Concentration) Physiology and Genetics Assay. The assay device consists of a plastic lid with ninety-six (96) pegs and a corresponding receiver plate with ninety-six (96) individual wells that have a maximum 200 μL working volume. Biofilm is established on the pegs under batch conditions (that is, no flow of nutrients into or out of an individual well) with gentle mixing. The established biofilm is transferred to a new receiver plate for disinfectant efficacy testing.3, 4 The reactor design allows for the simultaneous testing of multiple disinfectants or one disinfectant with multiple concentrations, and replicate samples, making the assay an efficient screening tool.  
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1.7 ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentioned in this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the risk of infringement of such rights, are entirely their own responsibility.  
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SCOPE
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5.1 This guide provides a protocol for detecting, characterizing, and quantifying nucleic acids (that is, DNA) of living and recently dead microorganisms in fuels and fuel-associated waters by means of a culture independent qPCR procedure. Microbial contamination is inferred when elevated DNA levels are detected in comparison to the expected background DNA level of a clean fuel and fuel system.  
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5.3 Just as the quantification of microorganisms using microbial growth media employs standardized formulations of growth conditions enabling the meaningful comparison of data from different laboratories (Practice D6974), this guide seeks to provide standardization to detect, characterize, and quantify nucleic acids associated with living and recently dead microorganisms in fuel-associated samples using qPCR.
Note 3: Many primers, an...
SCOPE
1.1 This guide covers procedures for using quantitative polymerase chain reaction (qPCR), a genomic tool, to detect, characterize and quantify nucleic acids associated with microbial DNA present in liquid fuels and fuel-associated water samples.  
1.1.1 Water samples that may be used in testing include, but are not limited to, water associated with crude oil or liquid fuels in storage tanks, fuel tanks, or pipelines.  
1.1.2 While the intent of this guide is to focus on the analysis of fuel-associated samples, the procedures described here are also relevant to the analysis of water used in hydrotesting of pipes and equipment, water injected into geological formations to maintain pressure and/or facilitate the recovery of hydrocarbons in oil and gas recovery, water co-produced during the production of oil and gas, water in fire protection sprinkler systems, potable water, industrial process water, and wastewater.  
1.1.3 To test a fuel sample, the live and recently dead microorganisms must be separated from the fuel phase which can include any DNA fragments by using one of various methods such as filtration or any other microbial capturing methods.  
1.1.4 Some of the protocol steps are universally required and are indicated by the use of the word must. Other protocol steps are testing-objective dependent. At those process steps, options are offered and the basis for choosing among them are explained.  
1.2 The guide describes the application of quantitative polymerase chain reaction (qPCR) technology to determine total bioburden or total microbial population present in fuel-associated samples using universal primers that allow for the quantification of 16S and 18S ribosomal RNA genes that are present in all prokaryotes (that is, bacteria and archaea) and eucaryotes (that is, mold and yeast collectively termed fungi), respectively.  
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SIGNIFICANCE AND USE
5.1 Bacteria that exist in biofilms are phenotypically different from suspended cells of the same genotype. Research has shown that biofilm bacteria are more difficult to kill than suspended bacteria (4, 5). Laboratory biofilms are engineered in growth reactors designed to produce a specific biofilm type. Altering system parameters will correspondingly result in a change in the biofilm. The purpose of this practice is to direct a user in the growth of a P. aeruginosa or S. aureus biofilm by clearly defining the operational parameters to grow a biofilm that can be assessed for efficacy using the Standard Test Method for Evaluating Disinfectant Efficacy Against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method (E2871).  
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SCOPE
1.1 This practice specifies the parameters for growing a Pseudomonas aeruginosa (ATCC 15442) or Staphylococcus aureus (ATCC 6538) biofilm that can be used for disinfectant efficacy testing using the Test Method for Evaluating Disinfectant Efficacy Against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method (E2871) or in an alternate method capable of accommodating the coupons used in the CDC Biofilm Reactor. The resulting biofilm is representative of generalized situations where biofilm exist on hard, non-porous surfaces under shear rather than being representative of one particular environment. Additional bacteria may be grown using the basic procedure outlined in this document, however, alternative preparation procedures for frozen stock cultures and biofilm generation (for example, medium concentrations, baffle speed, temperature, incubation times, coupon types, etc.) may be necessary.  
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5.1 This practice is to help in the development of protocols to assess the survival, removal and/or inactivation of human pathogens or their surrogates in indoor air. It accommodates the testing of technologies based on physical (for example, UV light) and chemical agents (for example, vaporized hydrogen peroxide) or simple microbial removal by air filtration or a combination thereof.  
5.2 While this practice is designed primarily for work with aerobic, mesophilic vegetative bacteria, it can be readily adapted to handle other classes of microbial pathogens or their surrogates.  
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SCOPE
1.1 This practice is to assess technologies for microbial decontamination of indoor air using a sealed, room-sized chamber (~24 m3) as recommended by the U.S. Environmental Protection Agency (3). The test microbe is aerosolized inside the chamber where a fan uniformly mixes the aerosols and keeps them airborne. Samples of the air are collected and assayed, firstly to determine the rates of physical and biological decay of the test microbe, and then to assess the air decontaminating activity of the technology under test as log10 or percentage reductions in viability per m3 (1). The air temperature and relative humidity (RH) in the chamber are measured and recorded during each test.  
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SIGNIFICANCE AND USE
5.1 In the battle to reduce medical device and implant-related infections, prevention of bacterial colonization of surfaces is a logical strategy. Bacterial colonization of a surface is a precursor to biofilm formation. Biofilm is the etiological agent of many implant and device-related infections and once established, microorganisms in biofilm can be up to 1000 times more tolerant to antibiotic therapy. Often the best treatment strategy is removal of the implant or device at a high socioeconomic cost. Catheter associated urinary tract infections (CAUTI) are the most prevalent of the device-related healthcare associated infections. Catheter associated infections account for 37 % of all hospital acquired infections (HAI) and 70 % of all nosocomial urinary tract infections (UTI) in the U.S. (2, 3). The Intraluminal Catheter Model (ICM) was developed to evaluate the ability of antimicrobial catheters to inhibit biofilm growth on the catheter lumen.  
5.2 The purpose of this test method is to direct a user in how to grow, sample, and analyze an E. coli biofilm in a urinary catheter under a constant flow of artificial urine. The test method incorporates operational parameters utilized in similar published methods (4). The E. coli biofilm that grows has a patchy appearance that varies across the catheter. Microscopically, the biofilm is heterogenous, with large clusters in some areas, and flat sheets of cells or even single cells in others. By 24 h, the biofilm is developed in the control catheters. If the goal is to monitor early stage biofilm development, then tubing and effluent samples need to be collected prior to the 24 h sample collection. Monitoring biofilm development requires sampling. The biofilm generated in the Intraluminal Catheter Model is suitable for comparison testing between antimicrobial and control catheters.
SCOPE
1.1 This test method specifies the operational parameters required to assess the ability of antimicrobial urinary catheters to prevent or control biofilm growth. Efficacy is reported as the log reduction in viable bacteria when compared to a repeatable (1)2  Escherichia coli biofilm grown in the intra-lumen of a urinary catheter under a constant flow of artificial urine.  
1.2 The test method is versatile and may also be used for growing and/or characterizing biofilms and suspended bacteria of different species, although this will require changing the operational parameters to optimize the method based upon the growth requirements of the new organism.  
1.3 This test method may be used to evaluate surface modified urinary catheters that contain no antimicrobial agent.  
1.4 This test method describes how to sample and analyze catheter segments and effluent for viable cells. Biofilm population density is recorded as log colony forming units per surface area. Suspended bacterial population density is reported as log colony forming units per volume.  
1.5 Basic microbiology training is required to perform this test method.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard  
1.7 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.  
1.8 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.

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