VETERINARY SERVICES MEMORANDUM NO. 800.117

TO: Veterinary Services Leadership Team

Directors, Center for Veterinary Biologics

Biologics Licensees, Permittees, and Applicants

FROM: John R. Clifford /s/ John R. Clifford, 8-12-2013

Deputy Administrator

SUBJECT: Guidance for Inactivation Studies

I. PURPOSE

This memorandum provides guidance for designing studies supporting the confirmation of 

the inactivation process for inactivated bacterial products and killed virus vaccines.

II. POLICY

Title 9, Code of Federal Regulations (9 CFR), section 113.200(a), states that for Killed Virus 

Vaccines “suitable tests to assure complete inactivation shall be written into the filed Outline 

of Production.” Further, 9 CFR 113.100(a)(1) states that “...an inactivated bacterial product 

shall be tested for viable bacteria and fungi as provided in [9 CFR]113.26.” The Center for 

Veterinary Biologics (CVB) interprets these regulations to mean that the process of 

inactivation must be consistent and supported by suitable studies. The appendix to this 

Memorandum describes a suitable approach for accomplishing this requirement.

III. IMPLEMENTATION/APPLICABILITY

Implementation is as of the date of this Memorandum. Guidance for Inactivation Studies is 

appended to this Memorandum. This guidance applies to products in the prelicense stage, 

except for those prelicense products whose inactivation procedures have been deemed 

satisfactory before the implementation date. The guidance also applies to licensed products 

whose manufacturing process or inactivation procedure is undergoing significant changes. 

This guidance does not apply to currently licensed products whose inactivation procedures 

remain unchanged. The guidance does not create new requirements; alternative approaches 

may be proposed.

Attachment: Appendix

United States 

Department of 

Agriculture

Animal and Plant 

Health Inspection 

Service

Veterinary Services

Washington, DC 

20250

 Safeguarding American Agriculture

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 An Equal Opportunity Provider and Employer 1-800-877-8339

 
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Appendix, Page 1

Appendix

Guidance for Inactivation Studies

1. Objective

These guidelines provide guidance for developing suitable studies and testing procedures 

to demonstrate that the inactivation procedure described in the manufacturer’s Outline of 

Production will consistently inactivate the infectious agent.

2. Background

2.1. Inactivation kinetics. Inactivation kinetics of microorganisms can only be directly 

observed up to the limit of detection (LOD) of the titration or assay method. Since 

practical inactivation endpoints are below the LOD, it is difficult to estimate the time 

until the targeted endpoint is achieved. Extrapolation of the inactivation curve to the 

endpoint is unsatisfactory for several reasons. The observed inactivation curves may 

be difficult to fit accurately due to their potentially multiphasic nature, and there are 

technical uncertainties in extrapolation itself. Most importantly, it is not unusual for 

the inactivation kinetics to change beyond the LOD. For practical reasons we 

describe the inactivation kinetics of a biological agent as composed of two stages: 

the first stage, when kinetics may be directly observed; and the second stage, when 

the rate of inactivation is not directly observed.

Goal. Since in practice it is difficult to directly determine the rate of inactivation in 

the second stage, one has to adopt a target intended to minimize the risk of infecting 

the vaccinated population and exposing non-vaccinates to residual live agent. A 

common target is less than one infectious unit per million doses (1x10-6 infectious 

units/dose), and that is the working target underlying these guidelines. Note, 

however, that it is not straightforward to relate the population risk to the target 

because of various uncertainties of several factors that vary widely between antigens, 

products, and lots, such as the number of viable organisms in an infectious unit and 

the volume of bulk antigen in the final vaccine dose. For that reason, the 

confirmation of inactivation testing performed on each vaccine lot is also an 

important part of the ongoing monitoring of the inactivation process.

3. Definitions

3.1. Inactivation Curve. The inactivation curve describes the relationship between the 

number of live particles and time of the inactivation process. The observed part (first 

stage) of the curve is the part obtained by conducting titrations at regular intervals 

after the start of inactivation. The unobserved part (second stage) of the curve is the 

part below the titration’s LOD. 
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3.2. Endpoint. The end of a sequence of five consecutive negative second-stage tests 

taken at least 1 hour apart.

3.3. Time to endpoint (TE). In a trial, the duration of exposure to the inactivation process 

to reach the endpoint.

3.4. Required time of inactivation (RTI). The required time of exposure to the inactivation 

process. The RTI is the maximum observed TE of at least three trials.

3.5. First stage assay. This is the titration or colony count testing procedure used to 

estimate the observed part of the inactivation curve and determine when the LOD is 

reached.

3.6. Second stage assay. This is a yes-or-no test that detects the presence or absence of 

the live agent at very low concentrations in a specified volume of material. It 

incorporates one or more concentration and amplification steps. In each trial, this 

binary assay is used during the second stage of the inactivation process to determine 

the TE. 

4. Study Design

Inactivation studies supporting the consistency of the inactivation procedures proposed in 

the Outline of Production must be conducted under conditions representative of actual 

production conditions. The guidance below represents practices the CVB considers 

valuable for evaluating the consistency and estimating the RTI, but it does not cover all 

possible scenarios. The CVB recommends that firms submit a draft protocol and 

preliminary data, if available, before initiating final studies.

4.1. Number of trials. Three trials.

4.2. Batch size. Two batches ≥10 percent; one ≥ 33 percent of the maximum production 

batch size. Data from smaller-scale batches used to identify and optimize the 

conditions for inactivation may be submitted as supporting data.

4.3. First-stage testing. The goal of first-stage testing is to characterize the observable 

part of the inactivation curve and determine the time when it reaches the LOD. 

Sampling should begin just before the start of the inactivation process and continue 

at regular intervals until the LOD is reached. Nine sample points are recommended, 

collected in duplicate. Include in the report the sample volume, and express the titer 

or count as per mL of the original culture, correcting for dilution and concentration 

steps. To maximize sensitivity a concentration step is recommended. Some 
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inactivation processes are very rapid, so it may not be feasible to have nine sampling 

points.

4.4. Second-stage testing. The goal of second-stage testing is to determine the TE of 

individual trials, and to use them to estimate RTI. Planning the initiation and timing 

of second-stage testing is based on preliminary studies, which assist in determining 

the observable and unobservable portions of the inactivation curve. Second-stage 

testing should begin approximately at the time when the first-stage testing reaches 

the LOD of the method. The TE is reached when five consecutive negative test 

results are obtained. These are performed in duplicate at least 1 hour apart using a 

minimum sample volume of 100 mL for most production procedures.

4.5. Inactivation conditions. Describe the inactivation conditions in detail. Some or all of 

the following conditions may be important for defining the inactivation process, 

depending on the inactivating agent and the microorganism. Additional parameters 

may be necessary to fully describe the process. 

4.5.1. Stage of culture. Conduct the inactivation studies on cultures harvested at the 

concentration (titer or count) and at the stage of growth specified in the 

Outline of Production. For purposes of validating the inactivation process, the 

concentration should be near the highest expected concentration described in 

the Outline of Production. 

4.5.2. pH. Conduct the inactivation studies at the pH specified in the Outline of 

Production. This is usually ± 0.2 pH unit.

4.5.3. Sample treatment and neutralization steps. Describe sample processing, 

including the concentration and neutralization steps. State how neutralization 

of the inactivation agent is determined.

4.5.4. Protein content. The goal is to have consistent conditions and minimize 

interference by extraneous protein. If the cells or virus particles are washed 

before treatment, indicate the washing steps. If a protein assay is used to 

establish the protein content, specify the range.

4.5.5. Temperature. Specify the temperature at which the inactivation procedure is 

conducted (± 2°C).

4.5.6. Osmolality. Specify the osmolality or describe the composition of the medium 

used during the inactivation process (e.g., twice-washed culture fluids were 

suspended in 0.15 M NaCl 0.05 M Na/K phosphate buffer pH=7.15). The goal 

is to have consistent conditions and minimize interference by variations in the 

osmolality of the medium.
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4.5.7. Conditions of exposure to the inactivating agent. The length of exposure to 

the inactivating agent should be specified in terms of concentration of the 

inactivating agent and time (hours and minutes) for the initial and any repeat 

exposures. Also, indicate adjustments of the medium pH, neutralization steps, 

and washing steps.

4.5.8. Facilities description. Describe the physical environment and equipment used 

for the inactivation process, so that it is consistent from batch to batch. This 

may include a description of the room and precautions taken to isolate it from 

production and finished product. Describe the incubator, inactivation vessels, 

(size, geometry), monitoring systems, mixing equipment, control of 

conditions, and if the fluids are transferred to a second or third vessel.

5. Summary

Preliminary test runs of the inactivation process using smaller batch sizes are essential to 

estimate conditions before confirming in larger batches. Confirm the consistency of the 

inactivation process in a minimum of three batches prepared according to the Outline of 

Production. Two batches must be ≥ 10 percent and the third ≥ 33 percent of the 

maximum batch size. Test for residual viability with a quantitative or quantal method 

(first stage) and follow with a yes-or-no test method (second stage). The first test method 

is used to show the initial kinetics of inactivation is consistent and to determine when 

testing with the second method should begin. The second test method is used to 

determine the endpoint and should incorporate concentration and amplification steps and 

replicate sampling of a minimum of 100 mL of culture. Sampling and testing continues at 

a minimum of 1-hour intervals until five negative tests in a row are obtained. The TE is 

the time of the fifth negative test and the RTI is the maximum TE for the three batches.

The 1-hour interval is a minimum; intervals may be wider.

6. Report

Submit a report that describes the inactivation process, the material and methods used in 

the study, and the limit of detection of the first- and second-stage testing. Summarize how 

the limit of detection was determined, the results of each trial, and the analysis of the 

results. The report should also include a detailed procedure for the confirmation of 

inactivation test method performed during manufacture of the product. This test should 

be very similar to the second-stage assay method in terms of sample size, concentration, 

and amplification steps. Submit all data in a useable format.
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7. Monitoring

The confirmation of inactivation testing of production serials will provide important

additional verification of the consistency of the inactivation procedure.