1、Designation: E2888 12Standard Practice forProcess for Inactivation of Rodent Retrovirus by pH1This standard is issued under the fixed designation E2888; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A n
2、umber 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 5 log10 inactivation of non-defective C-type retroviruses, which are endogenous to murinehybridoma and CHO cells a
3、nd are potentially present in theproduction stream of biopharmaceutical processes that userodent derived cell culture.1.2 The process parameters specified in this practice con-sistently assure 5 log10 inactivation of murine retrovirus byadjusting the pH of a process solution after initial affinityca
4、pture chromatography purification.1.3 This practice is applicable to mAb, IgG fusion, or otherrecombinant proteins produced from rodent cell lines (forexample, CHO or murine hybridoma), which do not targetretroviral proteins. Additionally, the low pH step is performedon a cell-free intermediate, pos
5、t initial capture using protein Achromatography.1.4 The 5 log10 inactivation of murine retrovirus claimedby using this practice will be utilized in conjunction with otherclearance unit operations (for example, chromatography andvirus retentive filtration) to assure sufficient total processclearance
6、of murine retroviruses, which will be supportive ofearly phase regulatory filings.1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.2. Terminology2.1 Definitions of Terms Specific to This Standard:2.1.1 IgG fusion proteina dim
7、eric protein comprised oftwo monomers, each monomer consisting of a peptide se-quence (usually a human receptor-like protein or proteinfragment) fused to the carboxyl-terminal of the Fc-domain of ahuman IgG antibody.2.1.1.1 DiscussionDimerization occurs by way of the Fcdomain.2.1.2 immunoglobulin G
8、(IgG)an antibody molecule com-posed of four peptide chains two heavy chains and twolight chains.2.1.2.1 DiscussionEach IgG has two antigen bindingsites. IgG constitutes 75 % of serum immunoglobulins inhumans. IgG molecules are synthesized and secreted by plasmaB cells. There are four IgG subclasses
9、(IgG1, 2, 3, and 4) inhumans, named in order of their abundance in serum (IgG1being the most abundant). Only human IgG1, IgG2, and IgG4show significant affinity to protein A.2.1.3 log10 reduction value (LRV)typically used to de-scribe the degree of reduction of a population, in this caserodent retro
10、virus, by the treatment process.2.1.3.1 DiscussionEach log reduction (10-1) represents a90 % reduction in the population. So a process shown toachieve a 6-log reduction (10-6) will reduce a population froma million (106)to1.2.1.4 monoclonal antibody (mAb)monospecific antibodieswhich have affinity fo
11、r the same antigen and are made from amaster cell bank, cloned from a parent cell.2.1.5 murine leukemia virus (MuLV)retroviruses namedfor their ability to cause cancer in murine (mouse) hosts.2.1.5.1 DiscussionMuLV is a member of the genus Gam-maretrovirus. MuLV is an enveloped spherical RNA viruswh
12、ich has a diameter of 80110 nm and has low chemicalresistance. MuLV is used as a model for non-defective C-typeendogenous retrovirus or retrovirus like particles produced bymurine hybridoma and CHO cell lines. MuLV is used to assessrodent retrovirus clearance of protein purification processesthat us
13、e rodent cells for production.2.1.6 recombinant proteinproduced from the expressionof recombinant DNA within living cells.2.1.6.1 DiscussionRecombinant DNA is genetically engi-neered by inserting foreign DNA into the DNA of an appro-priate host so that the foreign DNA is replicated along with thehos
14、t DNA.2.1.7 retrovirusan RNA virus that is propagated in a hostcell using the reverse transcriptase enzyme to produce DNAfrom its RNA genome.2.1.7.1 DiscussionDNA is then incorporated into thehosts genome by an integrase enzyme. The virus is thereafterreplicated as part of the host cells DNA. Retrov
15、iruses areenveloped viruses that belong to the viral family Retroviridae.1This practice is under the jurisdiction of ASTM Committee E55 on Manufac-ture of Pharmaceutical Products and is the direct responsibility of SubcommitteeE55.04 on General Biopharmaceutical Standards.Current edition approved Au
16、g. 1, 2012. Published September 2012. DOI:10.1520/E2888-12.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States12.1.8 modular validationa modular clearance study is onethat demonstrates virus removal or inactivation at individualsteps duri
17、ng the purification process (column chromatography,filtration, heat treatment, solvent/detergent treatment, low pHtreatment, etc.).2.1.8.1 DiscussionEach module, or unit operation, in thepurification scheme may be studied independently of the othermodules. Different model mAb may be used to demonstr
18、ateviral clearance in different modules, if necessary. If thepurification process of a product mAb differs at any of the virusremoval or inactivation modules from the model mAb, thismodule must be studied independently from the model. Theother, identical modules in the procedure may be extrapolatedt
19、o the product mAb. The total LRV of a purification processcan be obtained by adding the LRVs of the individual mod-ules.23. Significance and Use3.1 Rodent cell lines are widely used in the production ofbiological therapeutics such as monoclonal antibodies andrecombinant proteins. These cell lines ar
20、e known to containgenes encoding endogenous retroviral-like particles or toproduce infectious endogenous retrovirus. Despite the lack ofevidence for an association between such rodent retrovirusesand disease in man, the potential contamination of humantherapeutics poses patient safety concerns.Addit
21、ionally, adven-titious agents such as viruses can be introduced into a drugsubstance manufacturing process from other sources such asraw materials. Potential safety issues can be attributed to thesebiosafety testing for products made using rodent cell lines.33.2 Low pH inactivates retroviruses by de
22、naturing the viralenvelope proteins. Similar to all chemical reactions, thisdepends on reactant concentration (that is, H+ ion concentra-tion as measured by pH), time of reaction and temperature ofreaction. Implementing the parameters that give robust andeffective rodent retrovirus inactivation esta
23、blished by thispratice, in conjunction with other clearance unit operations (forexample, chromatography, virus retentive filtration) can assuresufficient purification process clearance of rodent retroviruses.43.3 This practice incorporates parameters that give robustand effective rodent retrovirus i
24、nactivation, which can be usedas modular validation for the low pH viral clearance moduleusing MuLV, the model non-defective C-type retrovirus endog-enous to murine hybridoma and CHO cells.4. Procedure4.1 For this practice, the primary variables specified are theconcentration range of buffer composi
25、tion elements, pH,temperature, protein concentration, and time during hold con-ditions.4.2 This practice will be applicable to mAb, IgG fusion, orother recombinant proteins, produced from rodent (forexample, CHO or murine hybridoma) cell lines, which do nottarget retroviral proteins.4.3 The low pH v
26、iral inactivation step must be performed ona cell-free intermediate, post initial capture using protein Achromatography.4.4 The inactivation process and the corresponding log10reduction of 5.0 are as follows:54.4.1 The hold temperature is 15C,4.4.2 The hold time is 30 minutes,4.4.3 The hold pH is 3.
27、6 throughout the course of the holdtime, and4.4.4 The buffer matrix will be glycine, citrate, or acetatebased, and the concentration of components will be in thefollowing ranges:4.4.4.1 If utilized, sodium chloride will be 500 mM inconcentration, and4.4.4.2 The protein concentration is 25 g/L.5. Key
28、words5.1 inactivation; retrovirus; rodentASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and th
29、e riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision o
30、f this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you should
31、make your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtaine
32、d by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).2U.S. Food and Drug Adminis
33、tration (FDA), Points to Consider in theManufacture and Testing of Monoclonal Antibody Products for Human Use, 1997,Department of Health and Human Services, Food and Drug Administration,Rockville, MD.3The International Conference on Harmonisation of Technical Requirements forRegistration of Pharmace
34、uticals for Human Use (ICH), Viral Safety Evaluation ofBiotechnology Products Derived from Cell Lines of Human or Animal Origin, Q5A,1999, Geneva, Switzerland.4Brorson, K., Krejci, S., Lee, K., Hamilton, E., Stein, K., et al., “BracketedGeneric Inactivation of Rodent Retroviruses by Low pH Treatment
35、 for MonoclonalAntibodies and Recombinant Proteins,” Biotechnology and Bioengineering , Vol 82,No. 3, 2003, pp. 321329.5Miesegaes, G., Bailey, M., Wilkommen, H., Chen, Q., Roush, D., et al.,“Proceedings of the 2009 Viral Clearance Symposium,” Journal of DevelopmentalBiology (Basel), Basel, Karger, Vol 133, 2010, pp. 2542.E2888 122
