1、Designation: E3042 16Standard Practice forProcess Step to Inactivate Rodent Retrovirus with TritonX-100 Treatment1,2This standard is issued under the fixed designation E3042; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year
2、 of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice assures effective inactivation of 4 log10ofinfectious rodent retrovirus (that is, reduction from 10
3、000 to 1infectious rodent retrovirus or removal of 99.99 % of infectiousrodent retroviruses) in the manufacturing processes of mono-clonal antibodies or immunoglobulin G (IgG) Fc fusion pro-teins manufactured in rodent-derived cell lines that do nottarget retroviral antigens. Rodent retrovirus is us
4、ed as a modelfor rodent cell substrate endogenous retrovirus-like particlespotentially present in the production stream of these proteins.1.2 The parameters specified for this practice areclarification, Triton X-100 detergent concentration, hold time,pH, and inactivation temperature.1.3 This practic
5、e can be used in conjunction with otherclearance or inactivation unit operations that are orthogonal tothis inactivation mechanism to achieve sufficient total processclearance or inactivation of rodent retrovirus.1.4 This detergent inactivation step is performed on aclarified, cell-free intermediate
6、 of the monoclonal antibody orIgG Fc fusion protein.1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsib
7、ility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Terminology2.1 Definitions of Terms Specific to This Standard:2.1.1 clarified, cell free intermediate, nin-process poollocated downstrea
8、m of the cell clarification unit operation(s),which should include a filtration step of 0.2 m nominal poresize, and upstream of the initial purification step in thepurification process of a monoclonal antibody or IgG Fc fusionprotein.2.1.1.1 DiscussionCell clarification unit operations areperformed
9、on the cell culture supernatant. Cell clarificationunit operations can be one or more of the following opera-tion(s): microfiltration, centrifugation, depth filtration, orflocculation, or combination thereof. The primary purpose ofcell clarification unit operation(s) is to remove cells used togenera
10、te monoclonal antibody or IgG Fc fusion protein andsome proportion of cellular debris from the cell culturesupernatant before the initial purification step. All clarificationsteps must include 0.2 m nominal pore size filtration tominimize the presence of virus aggregates, prior to detergentinactivat
11、ion. Freezing or prolonged storage between 0.2 mfiltration and detergent inactivation should be avoided.2.1.2 enveloped virus, nviruses in which the nucleic acidcomponent of the virus is surrounded by a lipid containingenvelope acquired from the host cell during virus assembly andbudding.2.1.2.1 Dis
12、cussionSome examples of enveloped virusesare from the families orthomyxoviridae (influenza), paramyxo-viridaemumps and measles, retroviridaehuman immunode-ficiency virus (HIV) and murine leukemia virus (MuLV), andherpesviridae human herpes virus (HHV), varicella-zostervirus (VZV), and pseudorabies v
13、irus (PRV).2.1.3 hold time, namount of time, after sufficient mixingtakes place, that the biological drug intermediate and retrovirusinteract with a specific chemical, in this case, the amount oftime the biological drug intermediate and retrovirus interactwith the Triton X-100.2.1.3.1 DiscussionDemo
14、nstration of sufficient mixing isthe responsibility of the manufacturer.2.1.4 immunoglobulin G, IgG, nantibody molecule com-posed of four peptide chainstwo gamma heavy chains andtwo light chains.2.1.4.1 DiscussionEach IgG has two antigen bindingsites. IgG constitutes 75 % of serum immunoglobulins in
15、humans. IgG molecules are synthesized and secreted by plasma1This practice is under the jurisdiction of ASTM Committee E55 on Manufac-ture of Pharmaceutical and Biopharmaceutical Products and is the direct responsi-bility of Subcommittee E55.04 on General Biopharmaceutical Standards.Current edition
16、approved Sept. 1, 2016. Published September 2016. DOI:10.1520/E3042-16.2Triton X-100 is a trademark of The Dow Chemical Company, Midlands,Michigan, http:/. The sole source of manufacture of the materialknown to the committee at this time is The Dow Chemical Company. If you areaware of alternative su
17、ppliers, please provide this information to ASTM Interna-tional Headquarters. Your comments will receive careful consideration at a meetingof the responsible technical committee,1which you may attend.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959.
18、United States1B cells. There are four IgG subclasses (IgG1, 2, 3, and 4) inhumans named in order of their abundance in serum (IgG1being the most abundant).2.1.5 immunoglobulin G (IgG) fusion protein, ndimericproteins comprised of two monomers, each monomer consist-ing of a peptide sequence (usually
19、a human receptor-likeprotein or protein fragment) fused to a human IgG antibody Fcdomain.2.1.6 effective viral clearance, na viral clearance unitoperation that removes or inactivates 4 log10reduction valueof virus.2.1.6.1 DiscussionInactivation requires a loss of infectiv-ity.2.1.7 log10reduction va
20、lue, LRV, nlog10reduction istypically used to describe the degree of reduction of anorganism population, in this case, rodent retrovirus, or otherenveloped virus, by the treatment process.2.1.7.1 DiscussionEach log10reduction represents a 90 %reduction in the organism population so a process shown t
21、oachieve a “6 log10reduction” will reduce a population from amillion organisms to one.2.1.8 modular viral validation, nmodular clearance studyis one that demonstrates virus removal or inactivation byindividual unit operations during the purification process(column chromatography, filtration, pasteur
22、ization, solvent/detergent, low pH, and so forth).2.1.8.1 DiscussionEach unit operation, or module, in thepurification scheme may be studied independently of the othermodules. Different model monoclonal antibodies (mAbs) maybe used to demonstrate viral clearance in different modules, ifnecessary. If
23、 the purification process parameters used in themanufacturing of a mAb product differs at any of the virusremoval or inactivation modules from the model mAb, thismodule shall be studied independently from the model. Theother, identical modules in the procedure may be extrapolatedto the product mAb.2
24、.1.9 monoclonal antibody, mAb, nmonospecific, recom-binant antibody manufactured using a production cell bank.2.1.10 murine leukemia virus, MuLV, nretrovirus namedfor its ability to cause cancer in murine (mouse) hosts.2.1.10.1 DiscussionMuLV is a member of the genusGammaretrovirus. MuLV is an envel
25、oped spherical RNA virusof 80 to 110 nm and has low chemical resistance. MuLV is usedas a model for C type endogenous retrovirus, retrovirus-likeparticles produced by rodent cell lines. MuLV, therefore, isused to assess retrovirus clearance of manufacturing processesthat use rodent cells for product
26、ion.2.1.11 inactivation temperature, ntemperature (C) of ma-trix in the container holding the Triton X-100 and the clarified,cell-free intermediate.2.1.12 retrovirus, nribonucleic acid (RNA) virus that ispropagated in a host cell using the reverse transcriptase enzymeto produce deoxyribonucleic acid
27、 (DNA) from its RNA ge-nome.2.1.12.1 DiscussionThe DNAis then incorporated into thehosts genome by an integrase enzyme. The virus thereafterreplicates as part of the host cells DNA. Retroviruses areenveloped viruses that belong to the viral family Retroviridae.2.1.13 Triton X-100 (polyethylene glyco
28、l p-(1,1,3,3-tetramethylbutyl)-phenyl ether), nnon-ionic surfactant; a liq-uid at room temperature.2.1.13.1 DiscussionTriton X-100 is also known as poly-ethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, oc-tyl phenol ethoxylate, Octylphenol Ethoxylate (non-ionic), andOctoxynol-9. The CAS nu
29、mber for Triton X-100 is 9002-93-1.In this practice, the chemical polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, CAS number 9002-93-1, willbe referred to as Triton X-100.2.1.14 Triton X-100 concentration, npercentage of TritonX-100 (% weight : volume) in the Triton X-100 detergentsol
30、ution.3. Significance and Use3.1 Rodent-derived cell lines are widely used in the produc-tion of biopharmaceutical drugs such as mAbs and Fc fusionproteins. These cell lines have been shown to contain genesencoding endogenous retroviral-like particles or endogenousretrovirus. Despite the lack of evi
31、dence for an associationbetween such rodent retroviruses and disease in humans, thepotential contamination of human therapeutics raises safetyconcerns for biopharmaceutical drugs. Additionally, adventi-tious agents such as viruses can be introduced into a biophar-maceutical drug substance manufactur
32、ing process from othersources, and potential safety issues can be attributed to thesepotential unknowns. For these reasons, effective viral clearanceis an essential aspect of an integrated approach combiningsafety testing and process characterization which ensures virussafety for biopharmaceutical d
33、rug products made using rodentcell lines.3.2 Solvent/detergent inactivation has been widely used fordecades to inactivate enveloped viruses in blood plasmaderived biopharmaceutical therapies (1-3).3Solvent/detergentsystems using the detergents Triton X-100 or Polysorbate 80along with the organic sol
34、vent tri(n-butyl)phosphate (TNBP)have been used to inactivate enveloped viruses by disruptingthe viral envelope thereby reducing the ability of the envelopedvirus to attach to and then infect the host cell (4 and 5).3.3 Most manufacturers of mAbs, recombinant proteins, andFc fusion proteins have foc
35、used on viral inactivation methodsusing the detergent Triton X-100 or Polysorbate 80 in theabsence of TNBP (6), which can interfere with subsequentbioprocessing steps. The ability of the detergents alone toinactivate retroviruses has been demonstrated in monoclonalantibodies produced in rodent-deriv
36、ed cell lines (6-9).Ata2011 workshop devoted to viral clearance steps used inbioprocessing (7), investigators from one firm showed incuba-tion with 0.2 % Triton X-100 for 60 min of hold time atambient temperature inactivated 5 log10of X-MuLV acrossfour separate mAbs in cell culture matrices.3The bol
37、dface numbers in parentheses refer to a list of references at the end ofthis standard.E3042 1623.3.1 At the same 2011 workshop (7), investigators from asecond firm confirmed that levels of protein concentration andlipid concentration had no observable effect on MuLV virusinactivation at levels of 0.
38、3 % Triton X-100.Additionally, eightdifferent monoclonal antibody Host Cell Culture Fluids(HCCF), were treated with 0.3 % Triton X-100 for a 60 minutehold time at 20C. Effective inactivation, 4 log10of inactiva-tion of MuLV virus, was seen for each antibody in theseexperiments.3.4 Quertinmont (8) de
39、monstrated that DNA level, totalprotein concentration, and lipid content (exceeding 1000 g/mL) in a 0.45 % (w/v) Triton X-100 detergent inactivation stepusing HCCF were not statistically significant to the detection ofMuLV virus following 60 minutes of inactivation using bothmonoclonal antibodies an
40、d Fc fusion proteins. Additionally,three Design of Experiment (DOE) robustness studies werecarried out for three separate molecules varying biological drugconcentration, total protein concentration, temperature, andTriton X-100 concentration. These studies demonstrated effec-tive viral inactivation
41、when Triton X-100 concentration is 0.2%, temperature is between 1525C, and hold time is 60minutes in HCCF.3.5 Blumel and Tounekti (9) showed complete inactivationof MuLV across 4 mAbs 2 IgGs and 2 immunoglobulin M(IgMs) for all time points (0, 5, 30, and 60 min) using 1.0 %Triton X-100 for a 60-minu
42、te hold time. The average logreduction factor (LRF) for these 15 studies was 3.89 log10.Analyses of the study data showed the higher level of TritonX-100 (1 %) necessitated a large dilution to mitigate cytotox-icity of the MuLV indicator cells. No detectable virus was seenat any of the time points t
43、ested across these 15 studies and theclaimed LRF was completely dependent on the starting viraltiter of the MuLV feed stock in these studies.3.6 The extent of this retroviral inactivation could bedependent on certain reaction parameters includingclarification, Triton X-100 concentration, hold time,
44、pH, andinactivation temperature. However, managing parameters thatgive robust and effective retrovirus inactivation as specified bythis practice, in conjunction with other clearance unitoperations, can assure effective retroviral inactivation.3.7 This practice incorporates parameters that give effec
45、tiveretrovirus inactivation, which can be used as modular valida-tion of the viral clearance process for the specified viruses.4. Procedure4.1 These specified parameters have been set to provideeffective viral reduction across a wide range of clarified cellculture matrices based on available data. H
46、owever, levelsoutside of these specified ranges, may provide effective viralreduction. Levels of reduction outside of these specifiedranges must be ensured by the manufacturer.4.2 For this practice, the key parameters specified areclarification, Triton X-100 detergent concentration, hold time,pH, an
47、d inactivation temperature.4.3 This practice is applicable to mAbs produced in rodent-derived cell lines in which the mAb or IgG Fc fusion Proteindoes not target a retroviral antigen.4.4 The inactivation process and the corresponding log10reduction value of 4.0 are as follows:4.4.1 This detergent in
48、activation step for this practice isperformed on a clarified, cell-free intermediate of the mAb orIgG Fc fusion protein. This clarification step must include 0.2m nominal pore size filtration to minimize the presence ofvirus aggregates, prior to detergent inactivation. Freezing orprolonged storage b
49、etween 0.2 m filtration and detergentinactivation should be avoided.4.4.2 The Triton X-100 concentration for this practice is0.5 %.4.4.3 The hold time for this practice is 60 min, followingsufficient mixing to ensure a homogenous distribution of TritonX-100 in the hold container.4.4.4 The pH range of the clarified, cell free intermediate forthis practice is 6.08.0.4.4.5 The reaction temperature range of the clarified, cell-free intermediate for this practice is 1525C.5. Keywords5.1 biological pharmaceutical drug
copyright@ 2008-2019 麦多课文库（www.mydoc123.com）网站版权所有
备案/许可证编号：苏ICP备17064731号-1