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VI.EVALUATION AND CHARACTERIZATION OF VIRAL CLEARANCE PROCEDURES Evaluation and characterization of due virus removal and/or inactivation procedures play an important role in establishing the safety of biotechnology products.Many instances of contamination in the past have occurred with agents whose presence was not known or even suspected,and though this happened to biological products derived from various source materials other than fully characterized cell lines,assessment of viral clearance will provide a measure of confidence that any unknown,unsuspected,and harmful viruses may be removed.Studies should be carried out in a manner that is well documented and controlled.
The objective of viral clearance studies is to assess process step(s)that can be considered to be effective in inactivating/removing viruses and to estimate quantitatively the overall level of virus reduction obtained by the process.This should be achieved by the deliberate addition (“spiking”)of significant amounts of a virus to the crude material and/or to different fractions obtained during the various process steps and demonstrating its removal or inactivation during the subsequent steps.It is not considered necessary to evaluate or characterize every step of a manufacturing process if adequate clearance is demonstrated by the use of fewer steps.It should be borne in mind that other steps in the process may have an indirect effect on the viral inactivation/removal achieved.Manufacturers should explain and justify the approach used in studies for evaluating virus clearance.
The reduction of virus infectivity may be achieved by removal of virus particles or by inactivation of viral infectivity.For each production step assessed,the possible mechanism of loss of viral infectivity should be described with regard to whether it is due to inactivation or removal.For inactivation steps,the study should be planned in such a way that samples are taken at different times and an inactivation curve constructed (see section VI.B.5.).
Viral clearance evaluation studies are performed to demonstrate the clearance of a virus known to be present in the MCBand/or to provide some level of assurance that adventitious viruses which could not be detected,or might gain access to the production process,would be cleared.Reduction factors are normally expressed on a logarithmic scale,which implies that,while residual virus infectivity will never be reduced to zero,it may be greatly reduced mathematically.
In addition to clearance studies for viruses known to be present,studies to characterize the ability to remove and/or inactivate other viruses should be conducted.The purpose of studies with viruses exhibiting a range of biochemical and biophysical properties that are not known or expected to be present is to characterize the robustness of the procedure rather than to achieve a specific inactivation or removal goal.Ademonstration of the capacity of the production process to inactivate or remove viruses is desirable (see section VI.C.).Such studies are not performed to evaluate a specific safety risk.Therefore,a specific clearance value need not be achieved.
A.The Choice of Viruses for the Evaluationand Characterization of Viral Clearance
Viruses for clearance evaluation and process characterization studies should be chosen to resemble viruses which may contaminate the product and to represent a wide range of physico-chemical properties in order to test the ability of the system to eliminate viruses in general.The manufacturer should justify the choice of viruses in accordance with the aims of the evaluation and characterization study and the guidance provided in this document.
1.“Relevant”Viruses and “Model”Viruses
Amajor issue in performing a viral clearance study is to determine which viruses should be used.Such viruses fall into three categories:“Relevant”viruses,specific “model”viruses,and nonspecific “model”viruses.
“Relevant”viruses are viruses used in process evaluation of viral clearance studies which are either the identified viruses,or of the same species as the viruses that are known,or likely to contaminate the cell substrate or any other reagents or materials used in the production process.The purification and/or inactivation process should demonstrate the capability to remove and/or inactivate such viruses.When a “relevant”virus is not available or when it is not well adapted to process evaluation of viral clearance studies (e.g.,it cannot be grown in vitro to sufficiently high titers),a specific “model”virus should be used as a substitute.An appropriate specific “model”virus may be a virus which is closely related to the known or suspected virus (same genus or family),having similar physical and chemical properties to the observed or suspected virus.
Cell lines derived from rodents usually contain endogenous retrovirus particles or retrovirus-like particles,which may be infectious (C-type particles)or noninfectious (cytoplasmic A-and R-type particles).The capacity of the manufacturing process to remove and/or inactivate rodent retroviruses from products obtained from such cells should be determined.This may be accomplished by using a murine leukemia virus,a specific “model”virus in the case of cells of murine origin.When human cell lines secreting monoclonal antibodies have been obtained by the immortalization of Blymphocytes by Epstein-Barr Virus (EBV),the ability of the manufacturing process to remove and/or inactivate a herpes virus should be determined.Pseudorabies virus may also be used as a specific “model”virus.
When the purpose is to characterize the capacity of the manufacturing process to remove and/or inactivate viruses in general,i.e.,to characterize the robustness of the clearance process,viral clearance characterization studies should be performed with nonspecific “model”viruses with differing properties.Data obtained from studies with “relevant”and/or specific “model”viruses may also contribute to this assessment.It is not necessary to test all types of viruses.Preference should be given to viruses that display a significant resistance to physical and/or chemical treatments.The results obtained for such viruses provide useful information about the ability of the production process to remove and/or inactivate viruses in general.The choice and number of viruses used will be influenced by the quality and characterization of the cell lines and the production process.
Examples of useful “model”viruses representing a range of physico-chemical structures and examples of viruses which have been used in viral clearance studies are given in Appendix 2and Table A-1.
Table A-1.Examples of Viruses Which Have Been Used in Viral Clearance Studies
2.Other Considerations
Additional points to be considered are as follows:
(a)Viruses which can be grown to high titer are desirable,although this may not always be possible.
(b)There should be an efficient and reliable assay for the detection of each virus used,for every stage of manufacturing that is tested.
(c)Consideration should be given to the health hazard which certain viruses may pose to the personnel performing the clearance studies.
B.Designand Implications of Viral Clearance Evaluationand Characterization Studies
1.Facility and Staff
It is inappropriate to introduce any virus into a production facility because of good manufacturing practice (GMP)constraints.Therefore,viral clearance studies should be conducted in a separate laboratory equipped for virological work and performed by staff with virological expertise in conjunction with production personnel involved in designing and preparing a scaled-down version of the purification process.
2.Scaled-down Production System
The validity of the scaling down should be demonstrated.The level of purification of the scaled-down version should represent as closely as possible the production procedure.For chromatographic equipment,column bed-height,linear flow-rate,flow-rate-to-bed-volume ratio (i.e.,contact time),buffer and gel types,pH,temperature,and concentration of protein,salt,and product should all be shown to be representative of commercial-scale manufacturing.Asimilar elution profile should result.For other procedures,similar considerations apply.Deviations that cannot be avoided should be discussed with regard to their influence on the results.
3.Analysis of Step-wise Elimination of Virus
When viral clearance studies are being performed,it is desirable to assess the contribution of more than one production step to virus elimination.Steps which are likely to clear virus should be individually assessed for their ability to remove and inactivate virus and careful consideration should be given to the exact definition of an individual step.Sufficient virus should be present in the material of each step to be tested so that an adequate assessment of the effectiveness of each step is obtained.Generally,virus should be added to in-process material of each step to be tested.In some cases,simply adding high titer virus to unpurified bulk and testing its concentration between steps will be sufficient.Where virus removal results from separation procedures,it is recommended that,if appropriate and if possible,the distribution of the virus load in the different fractions be investigated.When virucidal buffers are used in multiple steps within the manufacturing process,alternative strategies such as parallel spiking in less virucidal buffers may be carried out as part of the overall process assessment.The virus titer before and after each step being tested should be determined.Quantitative infectivity assays should have adequate sensitivity and reproducibility and should be performed with sufficient replicates to ensure adequate statistical validity of the result.Quantitative assays not associated with infectivity may be used if justified.Appropriate virus controls should be included in all infectivity assays to ensure the sensitivity of the method.Also,the statistics of sampling virus when at low concentrations should be considered (Appendix 3).
4.Determining Physical Removal Versus Inactivation
Reduction in virus infectivity may be achieved by the removal or inactivation of virus.For each production step assessed,the possible mechanism of loss of viral infectivity should be described with regard to whether it is due to inactivation or removal.If little clearance of infectivity is achieved by the production process,and the clearance of virus is considered to be a major factor in the safety of the product,specific or additional inactivation/removal steps should be introduced.It may be necessary to distinguish between removal and inactivation for a particular step,for example,when there is a possibility that a buffer used in more than one clearance step may contribute to inactivation during each step,i.e.,the contribution to inactivation by a buffer shared by several chromatographic steps and the removal achieved by each of these chromatographic steps should be distinguished.
5.Inactivation Assessment
For assessment of viral inactivation,unprocessed crude material or intermediate material should be spiked with infectious virus and the reduction factor calculated.It should be recognized that virus inactivation is not a simple,first order reaction and is usually more complex,with a fast “phase 1”and a slow “phase 2.”The study should,therefore,be planned in such a way that samples are taken at different times and an inactivation curve constructed.It is recommended that studies for inactivation include at least one time point less than the minimum exposure time and greater than zero,in addition to the minimum exposure time.Additional data are particularly important where the virus is a “relevant”virus known to be a human pathogen and an effective inactivation process is being designed.However,for inactivation studies in which nonspecific “model”viruses are used or when specific “model”viruses are used as surrogates for virus particles,such as the CHOintracytoplasmic retrovirus-like particles,reproducible clearance should be demonstrated in at least two independent studies.Whenever possible,the initial virus load should be determined from the virus that can be detected in the spiked starting material.If this is not possible,the initial virus load may be calculated from the titer of the spiking virus preparation.Where inactivation is too rapid to plot an inactivation curve using process conditions,appropriate controls should be performed to demonstrate that infectivity is indeed lost by inactivation.
6.Function and Regeneration of Columns
Over time and after repeated use,the ability of chromatography columns and other devices used in the purification scheme to clear virus may vary.Some estimate of the stability of the viral clearance after several uses may provide support for repeated use of such columns.Assurance should be provided that any virus potentially retained by the production system would be adequately destroyed or removed prior to reuse of the system.For example,such evidence may be provided by demonstrating that the cleaning and regeneration procedures do inactivate or remove virus.
7.Specific Precautions
(a)Care should be taken in preparing the high-titer virus to avoid aggregation which may enhance physical removal and decrease inactivation,thus distorting the correlation with actual production.
(b)Consideration should be given to the minimum quantity of virus which can be reliably assayed.
(c)The study should include parallel control assays to assess the loss of infectivity of the virus due to such reasons as the dilution,concentration,filtration or storage of samples before titration.
(d)The virus “spike”should be added to the product in a small volume so as not to dilute or change the characteristics of the product.Diluted,test-protein sample is no longer identical to the product obtained at commercial scale.
(e)Small differences in,for example,buffers,media,or reagents can substantially affect viral clearance.
(f)Virus inactivation is time-dependent;therefore,the amount of time a spiked product remains in a particular buffer solution or on a particular chromatography column should reflect the conditions of the commercial-scale process.
(g)Buffers and product should be evaluated independently for toxicity or interference in assays used to determine the virus titer,as these components may adversely affect the indicator cells.If the solutions are toxic to the indicator cells,dilution,adjustment of the pH,or dialysis of the buffer containing spiked virus might be necessary.If the product itself has anti-viral activity,the clearance study may need to be performed without the product in a “mock”run,although omitting the product or substituting a similar protein that does not have anti-viral activity could affect the behavior of the virus in some production steps.Sufficient controls to demonstrate the effect of procedures used solely to prepare the sample for assay (e.g.,dialysis,storage)on the removal/inactivation of the spiking virus should be included.
(h)Many purification schemes use the same or similar buffers or columns repetitively.The effects of this approach should be taken into account when analyzing the data.The effectiveness of virus elimination by a particular process may vary with the manufacturing stage at which it is used.
(i)Overall reduction factors may be underestimated where production conditions or buffers are too cytotoxic or virucidal and should be discussed on a case-by-case basis.Overall reduction factors may also be overestimated due to inherent limitations or inadequate design of viral clearance studies.
C.Interpretation of Viral Clearance Studies;Acceptability
The object of assessing virus inactivation/removal is to evaluate and characterize process steps that can be considered to be effective in inactivating/removing viruses and to estimate quantitatively the overall level of virus reduction obtained by the manufacturing process.For virus contaminants,as in Cases Bthrough E,it is important to show that not only is the virus eliminated or inactivated,but that there is excess capacity for viral clearance built into the purification process to assure an appropriate level of safety for the final product.The amount of virus eliminated or inactivated by the production process should be compared to the amount of virus which may be present in unprocessed bulk.
To carry out this comparison,it is important to estimate the amount of virus in the unprocessed bulk.This estimate should be obtained using assays for infectivity or other methods such as transmission electron microscopy (TEM).The entire purification process should be able to eliminate substantially more virus than is estimated to be present in a single-dose-equivalent of unprocessed bulk.See Appendix 4for calculation of virus reduction factors and Appendix 5for calculation of estimated particles per dose.
Manufacturers should recognize that clearance mechanisms may differ between virus classes.Acombination of factors should be considered when judging the data supporting the effectiveness of virus inactivation/removal procedures.These include:
(i)The appropriateness of the test viruses used;
(ii)The design of the clearance studies;
(iii)The log reduction achieved;
(iv)The time dependence of inactivation;
(v)The potential effects of variation in process parameters on virus inactivation/removal;
(vi)The limits of assay sensitivities;
(vii)The possible selectivity of inactivation/removal procedure(s)for certain classes of viruses.
Effective clearance may be achieved by any of the following:Multiple inactivation steps,multiple complementary separation steps,or combinations of inactivation and separation steps.Since separation methods may be dependent on the extremely specific physico-chemical properties of a virus which influence its interaction with gel matrices and precipitation properties,“model”viruses may be separated in a different manner than a target virus.Manufacturing parameters influencing separation should be properly defined and controlled.Differences may originate from changes in surface properties such as glycosylation.However,despite these potential variables,effective removal can be obtained by a combination of complementary separation steps or combinations of inactivation and separation steps.Therefore,well-designed separation steps,such as chromatographic procedures,filtration steps,and extractions,can be effective virus removal steps provided that they are performed under appropriately controlled conditions.An effective virus removal step should give reproducible reduction of virus load shown by at least two independent studies.
An overall reduction factor is generally expressed as the sum of the individual factors.However,reduction in virus titer of the order of 1log10or less would be considered negligible and would be ignored unless justified.
If little reduction of infectivity is achieved by the production process,and the removal of virus is considered to be a major factor in the safety of the product,a specific,additional inactivation/removal step or steps should be introduced.For all viruses,manufacturers should justify the acceptability of the reduction factors obtained.Results would be evaluated on the basis of the factors listed above.
D.Limitations of Viral Clearance Studies
Viral clearance studies are useful for contributing to the assurance that an acceptable level of safety in the final product is achieved but do not by themselves establish safety.However,a number of factors in the design and execution of viral clearance studies may lead to an incorrect estimate of the ability of the process to remove virus infectivity.These factors include the following:
1.Virus preparations used in clearance studies for a production process are likely to be produced in tissue culture.The behavior of a tissue culture virus in a production step may be different from that of the native virus,for example,if native and cultured viruses differ in purity or degree of aggregation.
2.Inactivation of virus infectivity frequently follows a biphasic curve in which a rapid initial phase is followed by a slower phase.It is possible that virus escaping a first inactivation step may be more resistant to subsequent steps.For example,if the resistant fraction takes the form of virus aggregates,infectivity may be resistant to a range of different chemical treatments and to heating.
3.The ability of the overall process to remove infectivity is expressed as the sum of the logarithm of the reductions at each step.The summation of the reduction factors of multiple steps,particularly of steps with little reduction (e.g.,below 1log10),may overestimate the true potential for virus elimination.Furthermore,reduction values achieved by repetition of identical or near identical procedures should not be included unless justified.
4.The expression of reduction factors as logarithmic reductions in titer implies that,while residual virus infectivity may be greatly reduced,it will never be reduced to zero.For example,a reduction in the infectivity of a preparation containing 8log10infectious units per milliliter (mL)by a factor of 8log10leaves zero log10per mLor one infectious unit per mL,taking into consideration the limit of detection of the assay.
5.Pilot-plant scale processing may differ from commercial-scale processing despite care taken to design the scaled-down process.
6.Addition of individual virus reduction factors resulting from similar inactivation mechanisms along the manufacturing process may overestimate overall viral clearance.
E.Statistics
The viral clearance studies should include the use of statistical analysis of the data to evaluate the results.The study results should be statistically valid to support the conclusions reached (see Appendix 3).
F.Reevaluation of Viral Clearance
Whenever significant changes in the production or purification process are made,the effect of that change,both direct and indirect,on viral clearance should be considered and the system re-evaluated as needed.For example,changes in production processes may cause significant changes in the amount of virus produced by the cell line;changes in process steps may change the extent of viral clearance.
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