Protocol for Evaluation of Virus Inactivation Using Low-pH Treatment
10.5731/pdajpst.2014.00969Access the most recent version at doi: 90-97
68, 2014 PDA J Pharm Sci and Tech
Dominick Vacante and Lisa Connell-Crowley
Treatment
Protocol for Evaluation of Virus Inactivation Using Low-pH
CONFERENCE PROCEEDING–APPENDIX
Protocol for Evaluation of Virus Inactivation Using Low-pH Treatment
DOMINICK VACANTE1,and LISA CONNELL-CROWLEY2
1Janssen Research&Development,LLC,Malvern,PA;and2Amgen Inc.,Seattle,WA?PDA,Inc.2014
Preface
Biopharmaceutical processes typically incorporate a dedicated procedure to inactivate retroviruses and other enveloped viruses susceptible to inactivation.A common inactivation procedure is low-pH hold,in which the product pool is titrated to pH?4.0with acid and held for a speci?c amount of time.Worse-case conditions for low-pH inactivation of retroviruses are now well known and include the upper limit of the target pH,lower limit of manufacturing temperature, and shortest hold duration.This protocol assembles current industry knowledge of considerations for per-forming well-controlled studies for assessment of vi-rus inactivation by low-pH inactivation,and includes guidance on modeling worse-case conditions based on manufacturing ranges.An example calculation of the log reduction value(LRV)using output virus titration data is also provided.
1.Purpose
The purpose of this protocol is to outline the equipment,procedures,and controls for assessing virus inactivation by low-pH treatment.The ki-netics of inactivation will be determined by eval-uating the virus titer at the start and end of the low-pH hold,and at one or more intermediate timepoints.
2.Scope
The scope of this document applies to monoclonal antibody products and other therapeutic protein products where virus inactivation is performed using a low-pH treatment.Viruses used for the virus inactivation assessment include murine leu-kemia viruses and other enveloped viruses(e.g., pseudorabies virus)that are susceptible to this form of inactivation.In addition,the principles of ICH Q5A guideline are applied.3.De?nitions
a.MuLV:murine leukemia virus,a gamma retro-
virus.Types typically used are amphotrophic,
ecotropic,or xenotropic.
b.Process pool:pool from the process that con-
tains product
c.LRV:log reduction value?log10(virus in/
virus out)
4.General Considerations
a.Study design
Assessment of viral inactivation should use pro-
tocols and procedures that are consistent with
the manufacturing-scale viral inactivation step
that is being modeled.This includes using rep-
resentative product process pools with a repre-
sentative product concentration,and compara-
ble pH titration and measurement procedures.
Additionally,virus inactivation should be as-
sessed using conditions that represent the
worst-case manufacturing conditions for inacti-
vation.These include targeting the upper end of
the low-pH target range and the shortest hold
time used in manufacturing.Also,the assess-
ment should be performed at the lower end of
the manufacturing temperature range because
the rate of inactivation decreases at lower tem-
peratures.Table I provides an example of man-
ufacturing conditions and worst-case conditions
for virus inactivation.
b.Virus assay
A validated virus assay that measures virus infec-
tivity should be used to determine the titer of
virus in the samples generated during the assess-
ment.Any cytotoxicity or interference of the sam-
ples with the selected assay should be mitigated
by dilution(see Section7).
doi:10.5731/pdajpst.2014.00969
90PDA Journal of Pharmaceutical Science and Technology
c.Virus spike percentage
The virus will be spiked into the process pool, typically at no more than10%of pool volume.
See Section7for considerations for the spiked process pool.
d.Documentation
For virus inactivation studies intended for health authority submission,protocols should be prepared for the virus assay and the low-pH treatment operation.All study documentation prepared by the virus testing lab,which includes virus assay protocols and results,should have quality unit oversight,under a written agreement that outlines critical quality expectations(quality agreement)if conducted by a third party.Operat-ing protocols for the low-pH treatment operation should have some form of quality oversight,de-pending on the product development stage.For
early-stage development,operating protocols may
be reviewed by the process sciences group and a
quality unit(or equivalent function)with an ap-
propriate science background,whereas late-stage
development operating protocols(phase3,pro-
cess validation,etc.),should have quality unit
oversight(1).In addition,a process record or
other form of documentation should be available
to record data and other information associated
with the study.Origin and composition of the
process intermediate used in the virus study
should be documented.
e.Precautions
Follow universal precautions and applicable
biosafety level when handling infectious virus
and samples containing infectious virus.
5.Responsibility
The product sponsor company is responsible for all procedures performed by their personnel and by contract testing company personnel.
6.Equipment and Materials
a.Equipment
i.pH meter
Because the low-pH inactivation study will be performed at a temperature lower than25°C, the pH meter used should have the ability to compensate for temperature.Depending on the meter,the temperature can be determined using a pH probe containing temperature probe or a sep-arate temperature probe that plugs into the meter.
Alternatively,some meters allow for a tem-perature to be entered in,and some have pre-programmed calibration settings.The pH me-ter should allow enough time for the probe to equilibrate in the solution before displaying the pH reading.A meter set up with a gel-?lled pH probe will need a longer time to equilibrate than a liquid-?lled pH probe.The pH meter should be on a regular calibration and maintenance schedule.
Note:Automatic temperature compensation (ATC)on the pH meter corrects only for the temperature effect on the pH probe itself.
ATC does not adjust for the actual shift in pH resulting from temperature changes within a given solution.
Table I
Example of Manufacturing Conditions and Worst-Case Conditions for the Virus Inactivation Study
Temperature
(°C)pH
Protein Concentration
(g/L)
Manufacturing a18–24 3.4–3.815–35
Virus inactivation
(worst-case
conditions)
17.5b 3.85c25d
a The manufacturing conditions in this example relate
to ranges that may be near the edge of failure.During
manufacturing,the operating ranges may be more
narrowly de?ned.
b The thermometer in this example has a variation of
0.5°C,therefore,the worst-case condition is per-
formed at17.5°C so the upper limit of the thermom-
eter is equal to the manufacturing lower limit of18°C.
The variation in the thermometer’s measurement
should be considered when designing the virus inac-
tivation study.
c In this example,the pH probe has a variation of
?0.05pH unit.Therefore,when set at3.85,the lower
limit is3.8,which is equal to the manufacturing upper
limit.The variation in pH probe measurement should
be considered when designing the virus inactivation
study.
d Protein concentration in this rang
e has minimal im-
pact on low-pH inactivation.Therefore,in this exam-
ple the mid-point is selected.
91 Vol.68,No.1,January-February2014
ii.pH probe:
The pH probe can be gel-?lled or liquid-?lled, and should be Tris buffer–compatible,as ap-propriate.The health of the probe can have a signi?cant impact on the pH measurement.
Contact with protein solutions over time can foul the glass pores of the probe and slow the passage of ions,leading to inaccurate measure-ment.The percent slope is not always a good indicator of poor probe health because an inac-curate probe can still have an acceptable slope.
An alternative measure of pH probe health is mV offset,which denotes the difference in mV between the reference electrode and the mea-suring electrode in pH7.0standard solution, which under ideal conditions should be zero.
Signi?cant deviation can indicate that the probe is fouled.If a probe is used for several studies, probe maintenance,calibration,and lifetime should be prospectively de?ned.Alternatively,
a new probe can be used for each study.
iii.Circulating water bath or equivalent equip-ment with temperature control set at the target
temperature
iv.Calibrated thermometer or thermocouple,to verify temperature of water bath
v.Balance and traceable weights,if measuring by weight
vi.Pipettors,calibrated and within calibration ex-piry
vii.Timer
b.Materials
i.pH standard solutions:Standards at pH7,pH4,
and others as appropriate.It is preferable to bracket the pH range or use a3point calibration wherein the third buffer has a pH value as close as possible to the target pH of the sample.
Standard solutions should be stored according to vendor recommendations and be within their expiry.
ii.Virus stock:
Information regarding the preparation of the virus stock is expected to be provided or de-scribed to the study sponsor.The virus stock
should be prepared from a controlled stock or source as outlined in PDA Technical Report47.
Ideally,master and working virus banks are es-tablished,if using a two-tiered banking system.
Methods of preparation are according to a stan-dard operating procedure.Tests for virus banks may be more extensive than those for virus pro-duction lots used in the spiking study.As outlined in PDA Technical Report47,virus production lots are tested for identity,sterility,and potency;
however,other tests may be applied.For exam-ple,purity tests can be useful to understand po-tential variability in inactivation when working at or near pH3.9where inactivation may be depen-dent on the matrix.The composition of the pro-cess intermediate should not signi?cantly be in-?uenced by the virus spike;this should be considered when using stabilizers in the virus spike preparations and/or in the virus spike per-centage.
Virus production lot testing:
a.Potency;titer
b.Purity;can include such attributes as ag-
gregation status,protein content,host cell
DNA/RNA,and particle-to-infectivity ra-
tio.Recommended,as appropriate
c.Safety;sterility or bioburden
d.Identity
e.Formulation
iii.Process pool:
The process pool is the starting material for the virus inactivation assessment.The process pool is provided by the product sponsor and should be representative of the manufacturing process.Prior to study start,the pool should be thawed(if required)and equilibrated to the testing temperature.
iv.Acid titrant:
The acid titrant(e.g.,acetic acid,citric acid, phosphoric acid)is used to lower the pH of the process pool to the target.The acid titrant should be the same acid as that used in the manufactur-ing process and should be formulated according to the manufacturer’s procedure.For some situ-
92PDA Journal of Pharmaceutical Science and Technology
ations,an option may be to use a higher molarity
acid titrant to lower the pH.
v.Base titrant:
The base titrant is used to neutralize low-pH
material(e.g.,1M HEPES,1M Tris base)in
order to stop the inactivation reaction at the
desired time prior to virus assay.It is good
practice to prevent pH overshoots,that is,
where the sample becomes strongly basic,by
using a weaker or dilute base.The base titration
is not intended to model the manufacturing
process.An alternative neutralization proce-
dure is dilution of the low-pH process pool
using cell culture medium followed by addition
of base titrant,if necessary.
vi.Cell culture medium or other negative control medium(optional):
The spiked cell culture medium control can be
used as an indication that the infectivity assay
is performing as expected.Additionally,it may
indicate whether the process pool was incor-
rectly neutralized or there is inherent instabil-
ity of the spiking virus in the neutralized pro-
cess pool from a non-pH-related interaction
between the virus and process?uid.
7.Preparation for the Virus Inactivation Study
a.Determine amount of acid titrant needed to reach
low-pH target
Given that virus preparations are formulated at neutral pH,the virus spike may increase the pH of the process pool,depending on the percent virus spike used and the properties of the pool.
Thus,the amount of acid titrant required to titrate the process pool to the low-pH target in the presence of the virus spike must be deter-mined in advance of the study.During the inac-tivation study,the pool will be titrated to a setpoint that,upon addition of the virus spike, results in the correct pH for the low-pH hold.
The virus formulation buffer(without virus)may be used as a replacement when handling of virus is prohibited.
b.Determine amount of base titrant needed to neu-
tralize low-pH pool
Prior to initiating the cytotoxicity and virus in-terference study(Section7c),the amount of base
titrant should be determined to return the low-pH process pool to a neutral pH suitable for cell culture;typically this is in the pH range of6.5–8.0.In some situations,the prod-uct or antibody at high concentration may not be soluble at neutral pH,and dilution in cell culture medium is used for neutralization.As noted,an alternative neutralization procedure is dilution of the low-pH process pool using cell culture medium followed by addition of base titrant,if necessary.
c.Cytotoxicity and virus interference testing
The neutralized,low-pH process pool must be evaluated for potential impact on the virus assay.
The pool can affect the assay by either being cytotoxic to the indicator cells or by interfering with virus infection of the cells.If diluted,the lowest dilution of the test article that does not result in cytotoxicity or interference should be used for the inactivation assessment study.Cy-totoxicity is typically evaluated as a percent-age of the cell culture affected(e.g.,25%, 50%,etc).
Viral interference may be determined by add-ing a?xed amount of virus to the neutralized low-pH process pool and calculating the recov-ery versus a medium control spiked with the same?xed amount of virus.Viral interference can also be determined by using dilutions of the neutralized low-pH pool to titer the virus.
Assay interference is determined by comparing the virus titer using the dilutions of the neu-tralized low-pH pool to the titer of the spiking virus control(titration assay positive control;
refer to Section8b).Due to the inherent vari-ation of virus titration assays,signi?cant ef-fects,in the range of0.5log10or more,are required to conclusively indicate the presence of viral interference.Also,consider that multiple serial dilutions of a high titer virus stock may introduce variability in the titer calculation.
8.Virus Inactivation Study Procedure
The low-pH inactivation study is expected to be performed in two independent runs according to the following procedure.Each step will be ini-tialed and dated as required in the applicable process record.The virus will be spiked into the process pool,typically at1%or5%,but not more than10%of pool volume.Controls will be used to monitor stability of the virus in the process con-
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Vol.68,No.1,January-February2014
ditions at neutral pH.See attachment 1for a ?ow chart and summary of samples.It is recommended to monitor the pH at the beginning (optional)and at the end of the inactivation hold time with a pH probe to ensure that the correct target pH has been reached upon the addition of virus,and remains at that pH for the duration of the inactivation.A timeline of the inactivation procedure is provided in Figure 1.
a.pH probe calibration
Calibrate pH probe using pH standard buffers per the pH meter manufacturer’s instructions.Best practice is to equilibrate the probe and buffers at the testing temperature prior to calibration.b.Control preparations for the virus inactivation study i.Spiking virus control (sample number 1in At-tachment 1):
As appropriate,pool vials of spiking virus into a common vessel.Aliquot a portion of the pooled virus and label as spiking virus control.Place on ice until titration.ii.Process pool control (sample numbers 3and 9in Attachment 1):
Equilibrate the process pool at the treatment temperature.Record temperature.Add acid titrant to the process pool as required to reach target pH (Section 7a),then neutralize for the cell culture assay as outlined in Section 7b.Add spiking virus into the neutralized low-pH process pool and mix.Record time and temperature.Remove the T ?0sample and place on ice until titration.This is sample num-ber 3in Attachment 1.
Incubate the remaining spiked neutralized pro-cess pool until T ?end time of treatment at the treatment temperature.Record time and tem-perature.At that time,place samples on ice until titration.This is sample number 9in At-tachment 1.
iii.Cell culture medium control,optional (sample
numbers 2and 8in Attachment 1):
Equilibrate cell culture medium at the treat-ment temperature.Record temperature.
Add spiking virus into cell culture medium and mix.Record time.After spiking,remove the T ?0sample and place on ice until titration.This is sample number 2in Attachment 1.Incubate the remaining spiked cell culture me-dium until T ?end time of treatment at the treatment temperature.Record time and tem-perature.At that time,remove sample and place samples on ice until titration.This is sample number 8in Attachment 1.
Equilibrate process pool at target temperature, pH probe calibra?on
Spiked process pool control,
T=0 (Sample 3)
Spiked cell culture medium,
T=0 (Sample 2)
Thaw spiking virus
(Sample 1)
Spiked process pool control,
T=end (Sample 9)
Spiked cell culture medium,
T=end (Sample 8)
Low pH,T=0 (Sample 4)Low pH,
T=intermediate ?me 1
(Sample 5)Low pH,
T=intermediate ?me 2
(Sample 6)Low pH, T=end (Sample 7)
Figure 1
Timeline of the low-pH inactivation procedure.The sample numbers in the timeline refer to the samples outlined in Attachment 1.Although four low-pH timepoints are presented in this example,multiple samples may be taken to demonstrate the inactivation kinetics and/or to establish the lower and upper limits for the low-pH inactivation procedure.
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PDA Journal of Pharmaceutical Science and Technology
c.Inactivation treatment of the process pool(sam-
ple numbers4,5,6,and7in Attachment1)
Note:The following is an example of an inacti-vation procedure;alternative approaches are ac-ceptable.
i.Equilibrate process pool and acid titrant at the
appropriate temperature in water bath.Record temperature.Ensure there is suf?cient pool mate-rial for all samples and sample pH measurements. ii.If appropriate,adjust pH to target pH using acid titrant,taking into account the impact of the sub-sequent virus spike on the?nal pH.Measure pH.
Record pH and sample volume or weight.
iii.Add spiking virus and mix.Measure pH and correct pH if needed to lower pH to
target.
Note:If the pH measurement of the spiked
low-pH process pool is below the target pH,
the study is invalid and should be re-started.
Record pH,pool volume or weight,volume of
spiking virus added and time;this is T?0.
Immediately remove samples,neutralize and la-
bel as T?0(sample number4in Attachment1).
Record pH,sample volume,and time.
Neutralize per procedure(Section7b).
Place samples on ice until titration.This is sam-
ple number4in Attachment1.
iv.Incubate virus-spiked process pool at target temperature.Record temperature.
v.At T?intermediate time1,2,and T?end time:
Remove sample.Record pH,sample volume, temperature,and time.
Neutralize per procedure(Section7b).
Place samples on ice until titration.These are sample numbers5,6,and7in Attachment1.
d.Analysis
The selected virus infectivity assay is used to determine the virus titer of samples and con-trols.The LRVs are calculated using virus titer of the spiked(neutralized)process pool con-trol after treatment time,T?end(sample number9in Attachment1)as the load or virus in.If this titer is signi?cantly different from the virus titer of the spiked(neutralized)pro-cess pool control at T?0(sample number3in
Attachment1),this should be considered in the interpretation of the test results;it can invali-date the assessment.The virus titer at each timepoint(virus out samples)should be used to determine the inactivation kinetics and LRV.Typically,the lower LRV of the two independent low-pH runs is reported for the inactivation procedure LRV for the overall process viral clearance.
The following is an example of an LRV cal-culation for a low-pH inactivation study with-out a statistical calculation for con?dence in-terval.Because there is no change in volume of the low-pH inactivation step during manu-facturing,there should not be a contribution to the LRV from a change in processing volume from virus in to virus out.The example uses a virus infectivity assay with results reported as focus forming units(FFU):
i.Preparation of low-pH process pool:
40mL process pool?4.0mL acid titrant?44 mL total low-pH process pool
ii.Load or virus in:
Neutralized low-pH process pool spiked with virus at T?end time(sample number9in Attachment1)
Volume?3mL low-pH process pool?0.6mL base titrant?0.3mL virus
?3.9mL total for titration
Titer?1?106FFU/mL
Load or virus in?1?106FFU/mL?3.9mL (total pool)?3.3mL(low-pH process pool plus virus)x(Sample dilution,if performed,in this example?1)
?1.2?106FFU/mL
iii.Virus out:
Low-pH pool spiked with virus at T?intermedi-ate time1(sample number5in Attachment1)
Volume of spiked pool?30mL low-pH process pool?3mL virus
?33mL total(extra volume to allow for pH mea-surements)
Remove3mL for titration and add0.6mL base titrant?3.6mL total for titration
Titer??1x101FFU/mL
Virus out??1?101FFU/mL?3.6mL?3mL (to correct for dilution for adding base titrant)?(Sample dilution,if performed,in this example?1)?1.2?101FFU/mL
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Vol.68,No.1,January-February2014
iv.LRV calculation:
LRV?log10(virus in/virus out)
?log10(1.2?106FFU/mL??1.2?101 FFU/mL)
?5.0
e.Acceptance criteria are as follows:
i.There is no minimum acceptable viral clear-
ance
ii.The virus spiking assessment is expected to be performed in two independent runs and should provide similar inactivation kinetics
iii.The medium control,if performed,and process pool control should show no more than1.0log
decrease from T?0to T?end time,unless
justi?ed
iv.The positive and negative control acceptance criteria for the virus titration assay must be valid Acknowledgments:
The authors thank the following individuals for in-valuable feedback and contributions to this protocol: Bob Steininger(Acceleron),David Farb(Macrogen-ics),David Roush(Merck),Dayue Chen(Eli Lilly), Eileen Wilson(GSK),Hannelore Willkommen(RBS consulting),Jeff Ucran(Acceleron),Judy Glynn (P?zer),Kurt Brorson(CDER/FDA),Lenore Norling (Genentech),Masahiro Fukunaga(Chugai),Matthew Dickson(Medimmune),Olga Galperina(HGS),Rich-ard Chen(Imclone),Scott Tobler(Merck),Shohei Kobayashi(Chugai).
9.References and Related Documents
1.Food and Drug Administration(FDA).Guidance for
industry.Process validation:general principles and practices.FDA:Bethesda,MD,January,2011. 2.International Conference on Harmonisation
(ICH).Viral safety evaluation of biotechnology
Attachment1.
Flow Chart of Samples for the Low-pH Viral Inactivation Study
Sample
Number Sample Name Control Low-pH Treatment Purpose
1Spiking virus(may also be the titration assay positive
control)Spiking virus Determines if the assay is
performing as expected
2Medium control,T?0Cell culture medium spiked
with virus(T?0)Provides information on the expected virus titer without the process pool at T?0
3Process control,T?0Neutralized low-pH process
pool spiked with virus(T?
0)Provides the expected virus titer in the presence of the process pool at T?0
4Low-pH pool,T?0Low-pH process pool,spiked
with virus(T?0)Provides information if immediate inactivation of virus occurs in the process pool low-pH conditions
5Low-pH pool,T?
intermediate time1Low-pH process pool,spiked
with virus(T?
intermediate time1)
Virus out sample
6Low-pH pool,T?
intermediate time2Low-pH process pool,spiked
with virus(T?
intermediate time2)
Virus out sample
7Low-pH pool,T?end time Low-pH process pool,spiked
with virus(T?end time)
Virus out sample
8Medium control,T?end
time Cell culture medium spiked
with virus(T?end time)
Provides information on the
stability of the virus
throughout the inactivation
procedure
9Process control,T?end time Neutralized low-pH process
pool spiked with virus(T?
end time)Load or virus in sample.Also, provides information on the stability of the virus in the presence of the process pool throughout the inactivation procedure
96PDA Journal of Pharmaceutical Science and Technology
products derived from cell lines of human or animal origin,ICH Q5a(R1),23September1999.
3.Guideline on virus safety evaluation of biotech-
nological investigational medicinal products, EMEA/CHMP/BWP/398498/2005,1February 2009.4.FDA.Points to consider in the manufacture and
testing of monoclonal antibody products for hu-man use.FDA,February28,1997.
5.Parenteral Drug Association Technical Report47.
Preparation of Virus Spikes Used for Virus Clear-ance Studies,2010.
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