Potency Test References

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POTENCY TEST REFERENCES

Relevant regulations and guidance documents:

Title 9, Code of Federal Regulations (9CFR 101.5, 113.8, 113.XX series containing Standard 

Requirements 

Veterinary Services Memorandum (VSM): 800.90 Guidelines for Veterinary Biological Relative 

Potency Assays and Reference Preparations Based on ELISA Antigen Quantification 

VSM 800.112 Guidelines for Validation of In Vitro Potency Assays

VSM 800.118 Live Master References

VSM 800.211 Guidelines for Master Reference Qualification and Requalification

Reviewers manual

4_4_3_MasterReferenceQualificationRequalificationandUnderstandingVSM800211

Contents 

Master reference description

Working references and qualifying serials

Master reference qualification and requalification

Temporary reference dating extensions

1. Master Reference Description

The Master Reference (MR) is a preparation of a biological product, made by the licensee, and 

derived from the Master Seed. The potency is correlated directly or indirectly to host animal 

immunogenicity (efficacy). The MR is used to evaluate the potency of serials of inactivated 

products by comparing the response of the serial to that of the MR in the potency test method. A 

MR may be fully formulated product (a bacterin, vaccine, antiserum, toxoid, immunomodulator), 

a purified preparation of the immunogen or antigen, or a non-adjuvanted harvested culture of 

microorganisms (live (see VSM 800.118) or inactivated) prepared according to the filed Outline 

of Production (9 CFR 101.5). In contrast, inactivated products with Standard Requirements (e.g. 

9CFR 113.120) that describe a Standard Reference refer to a preparation provided by CVB that 

has been assessed for potency usually by in-house animal testing. 

The potency test methods may be analytical or comparative (VSM 800.112 Appendix III) hence 

they may be quantitative or relative (response based). Regardless, the MR is treated the same.

The MR must be identified in the Outline of Production (not the Special Outline) by lot number 

and assigned an expiration date. The dating period begins on the date of the initiation of the 

efficacy study (date of first vaccination rounded to the end of that month) while the duration is 

set by one of several methods. 

In the past, the dating period for a MR was the same as the dating of the product or if the MR 

was frozen and the firm demonstrated the freezing and subsequent thawing did not affect the 

potency estimation (dose-response curves superimposable and magnitude same for never frozen 

and frozen thawed samples) the MR was given 5 years dating (9CFR 113.8(d)(2)). This view of 

MR expiration dating is being supplanted by the guidelines in VSM 800.211 which permits the 

expiry period to be as much as 15 years depending on several factors such date of licensure, 

validation of the potency assay and approval of the stability monitoring program (see VSM 
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800.211, 800.112 appendix III, and the reviewers manual 4.4.3. for the details). Note that the 

terms of use and requirements for requalification of the MR for products licensed before January 

1, 2011, (Previously Licensed Products or Legacy Products see VSM 800.211) are treated 

different than those licensed after that date.

The MR may be a concentrate

which are diluted in the appropriate matrix for use in the potency test method.

MR’s ideally would represent the minimum protective dose (MPD) of the product, but in 

practice they represent a dose shown to be protective in studies conducted by the firm and may 

be greater than the true minimum needed for protection. Thus, relative potency measurements 

should not be interpreted as direct measures of efficacy because two different antigen 

preparations may be equally effective in the immunogenicity trial but different in protective 

antigen content. In this scenario the animal challenge studies cannot evaluate the stability of the 

MR and only indicate that its current potency is at least the MPD. The initial potency of a MR 

may have been much greater than the true MPD, and the amount of degradation is not detected in 

the immunogenicity study because the “potency” is at least the MPD.

Note that not all inactivated products are tested by comparison with a MR. Some products are 

tested for potency in vivo and rely on clinical or serologic outcomes without a reference or they

follow a Standard Requirement as mentioned above. 

In addition to the MR, other biological preparations are usually run concurrently with a test serial 

for purposes of determining if the particular assay run is valid (i.e. not a “No Test”). These are 

usually called “positive controls” instead of references, but sometime firms will use these terms 

(as well as “standard”) interchangeably. Evaluate the context used and encourage clear 

terminology. Positive and negative controls or validity standards are not subject to the same 

scrutiny as MR but they should be lot controlled and have criteria for acceptance identified in the 

Outline of Production. 

2. Working Reference Description

The Working Reference (WR) is defined in 9CFR 101.5(p) “is the reference preparation that is 

used in the in vitro test for the release of serials of product. Working References may be:

MR or serials of product that have been prepared and qualified, in a manner acceptable to APHIS 

for use as reference preparations.” The WR is used in place of the MR and has a potency that is 

equal to or slightly greater than the MR. In terms of characteristics it is almost always product 

like and the dose response curves are parallel to the MR otherwise it cannot be used. The WR is 

used instead of the MR because it can be replenished reducing the depletion rate for the MR

Note

In relative potency (RP) assays the release and throughout dating level has been an RP≥1.0 and 

1.0 was defined as in the range 0.95-1.05. This is inconsistent with the treatment of live 

products which require an overage for assay variability, production variability and stability. In 

addition it is not consistent with our approach to assay validation which requires assessment of 

precision, accuracy, and ruggedness of the testing method. Draft documents addressing this are 

in process and will very likely modify the release and throughout dating value for relative 

potency assays as well as the quantitative in vitro assays for inactivated products.
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stocks. The WR has an expiry period identical to the product, regardless of the dating for the 

MR. The WR reference is determined to be suitable for use by comparing it to the MR in the 

approved potency assay. Hence the working reference is “qualified” by comparison to the MR in 

the potency assay. This means the MR must be unexpired and therefore qualified (at some point 

in time) by testing in an immunogenicity study. 

The WR used in relative potency assays usually has an RP-value >1.0 however it represents the 

MR and is evaluated as if it has an RP=1.0. No correction is permitted. This is in contrast to 

WR for quantitative assays where the WR is used to prepare a standard curve for extrapolation of 

the values for a serial. In this case the actual value of the WR is used.

3. Qualifying Serial Description

The Qualifying Serial (QS) is defined in 9CFR 101.5(q) and is a serial of the product that has a 

relative potency ≤ 1.0. This is used to qualify (in an immunogenicity study) the MR usually 

when the MR is not product like.

4. Internal control-see 800.112 App. I

5. Reference-see 800.112 App. I 

6. Standard-see 800.112 App. I 

7. Reagent Blank-see 800.112 App. I 

8. Unknown-see 800.112 App. I 

9. Live Master Reference-see 800.118 

10. Reference Standard-see 800.90 

11. Relationship between Master References, Working References, and Qualifying 

Serials

a. MR is the QS and the WR

The MR is a fully formulated serial of product ( “product-like Master Reference”). It is used to 

qualify itself and therefore is the QS and it is used in the potency assay for serial release so it is 

also the WR.

b. MR is not product like

The MR is either a purified or semi-purified preparation, concentrate or non-adjuvanted harvest 

fluids (e.g. Live Master Reference or inactivated fluids) that is not suitable for use in a 

qualification study because it is not product-like. In order to qualify this type of MR the firm 

prepares a QS that has a potency less than or equal to the MR or a designated dilution of the MR. 

The QS is evaluated in an efficacy study and if satisfactory the MR is qualified. 

• The MR may be used as the WR at the dilution used to measure the potency of the QS. 


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c. A summary of the firm’s efforts to qualify a reference. The summary should be 

provided when the firm is requesting an extension because of difficulties 

associated with conducting a study. What you as a reviewer have to determine is 

whether or not the firm is making a good faith effort or not. The firm must 

provide a summary of their efforts and address how they intend to solve the 

problem(s) encountered. 

2. Data. For “data acceptable and approved by APHIS,” firms have typically provided 

historical data, usually OD’s of the reference and/or plate controls, over the lifetime of 

the reference. If complete plate data were provided (including serial OD’s) such data 

would be useful as a crude tool to monitor the stability of the assay system as a whole

(note this is allowed in VSM 800.211 for previously licensed products). Note, however, 

that almost none of the currently approved relative potency tests are designed to provide a 

sensitive assessment of the stability of the reference. Rigorous evaluation of the stability 

of a reference depends on tests that measure its qualitative and quantitative characteristics 

independently of the potency test. OD values or parameter estimates from the potency 

test are not sufficient by themselves to determine if the reference is stable.

Independent testing may be performed on either the reference or an internal control as 

long as the internal control is assayed concurrently in the potency test. The independent 

testing needs to be performed with validated assays that measure one quantitative and 

qualitative characteristic other than the one(s) measured in the potency test. Monitoring 

these characteristics over time should provide sufficient information to evaluate if the 

reference and the potency test are performing satisfactorily.

For example, if the ELISA potency test is an indirect antigen capture sandwich assay 

using a monoclonal detecting antibody, the independent assays could be SDS-PAGE and 

Western Blotting where molecular size, antibody binding, and quantity (densitometry or 

precision dilution to extinction) are assessed. But note that because of the differences in 

products there is not a simple answer or one-size fits all approach. Consult with others 

with expertise in the product and testing methods.