Title: Proposals for Estimating Relative Potency
Release Date: 25 Feb 2023 Author: a Document Number: CVB-WI-0188.03
CONTROLLED//PROPIN//BASIC

Specifying a pseudo-linear region

Relative potency assays evaluated by the parallel line method must identify the pseudo-linear
regions of the two response curves that will be used for calculating the RP. To properly do that,
the assay must be optimized well enough to perform consistently. If so, it is usually easy to
specify a method for identifying the correct regions. The definition of relative potency as the
ratio of equipotent dilutions leads naturally to a method based on the response (OD), but the
structure of a serial release test may lead to a method that utilizes the dilutions, or both OD and
dilutions. Examples:

OD. Choose an OD response range based on supporting data to select the region where the
responses of reference and test serials are expected to behave linearly. Use all dilutions within
that range. Selection should be based on mean OD response at each dilution.

OD and dilutions. Choose a dilution range for the reference based on supporting data to select
the region where the response of the reference behaves linearly. Note the OD range spanned by
those dilutions. Then select the dilutions of the test serial that fall within that OD range.
Selection should be based on mean OD response at each dilution.

Dilutions. Choose dilution ranges based on supporting data to select the regions where the
responses of reference and test serials are expected to behave linearly. Because this method is not
primarily based on the response, it may be less consistent in capturing the appropriate regions, so
optionally a small amount of leeway in adjusting the selected dilution subset would be
acceptable.

e A single dilution shift of a subset within the candidate range is acceptable. For example,
if a candidate range for a given preparation is defined as a set of n consecutive dilutions,
then segments of length n-1 or n within that range may be considered for the final
choice.”

e The procedure for choosing the subsets from within the candidate range must be fully
specified and reproducible.’

e The pseudo-linear regions for the reference and serial need not use the same dilutions.
However, the OD response of the two preparations should overlap. A lack of such
overlap should result in declaring the test invalid.

? When this criterion is applied to a reference and test serial together, up to 9 possible combinations of segments
may be considered. For instance, choose at least 3 consecutive dilutions between dilutions 2 through 5. The allowed
segments are dilutions 2-4, 3-5, and 2-5. Avoid specifying that only segments of exactly n-1 dilutions (excluding
segments of length n) would be considered.

3 For instance, select the combination whose slope ratio is closest to 1.0.

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Title: Proposals for ae Potency
Release Date: 25 Feb 2023 Author: Document Number: CVB-WI-0188.03
CONTROLLED//PROPIN//BASIC

Parallel Line

Full Dose Response

Both a (nonzero) minimum slope and an

acceptance range for the ratio of the slopes of

the serial to the reference.

e Minimum slopes less than 0.10 in
minimum absolute value should not
be entertained. This can be used as a
default value.

e For transitioning assays only, an
acceptance range for the slope ratio
of 0.80 to 1.25, inclusive, will be
allowed for consistency with RelPot.

e For new assays, an acceptance range
tied to data, which includes 1.0 and
whose limits do not exceed 0.80 to
1.25, should be specified.! Proposals
should avoid using 0.80-1.25 as
default validity criteria.

Acceptance ranges for the ratios of upper
asymptote and scale factor between the
serial and the reference. Preferably these
ranges should be based on data and should
include 1.0. Proposed ranges exceeding
0.80 to 1.25 will not be entertained
without scientific justification. However,
proposals should avoid using 0.80-1.25 as
default validity criteria.

Specify the procedure for identifying the
pseudo-linear region. Specific guidance for
this option is given in the next section.

If an assay has a demonstrated “hook
effect” (an initial deviation from the upper
asymptote), it is acceptable to exclude the
dilutions involved in the hook effect

Acceptance ranges for positive and negative controls and reagent blanks

Software version number, and any settings and options that need to be specified.
e  Itis usually helpful for the firm to submit an electronic file illustrating the intended
calculations in the native format of the chosen software. This will help the Statistics
Section evaluate the suitability of the proposed method
e The phrase “version XXX or later” may be used for common curve fitting software
with transparent algorithms, such as SoftMax or GenS. It does not apply to software

based on undocumented proprietary algorithms that may change at vendor
discretion or software used for other purposes.

Auxiliary files, such as template files, should be version controlled and specified.

+ One option for designing such an interval would be to calculate the mean + two standard deviations of slope ratios
from assay validation data. Of the two resulting limits, use the one furthest from 1.0, and reciprocate it to form the
corresponding other limit. This method creates an interval symmetric about the ideal slope ratio of 1.0 but tied to the
variability of the observed data.

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Title: Proposals for Estimating Relative Potency
Release Date: 25 Feb 2023 Author: — Document Number: CVB-WI-0188.03
CONTROLLED//PROPIN//BASIC

Pre-dilution

For Outlines previously approved for pre-dilution of serials under various scenarios, CVB-PEL
strongly recommends that firms consider adopting either the OD or OD and dilutions methods
(described above) to specify the pseudo-linear region. Calculations made using these approaches

are reproducible, even when the option to pre-dilute may result in different starting dilutions
among serials.

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Release Date: 25 Feb 2023 Author:

Veterinary Services

Center for Veterinary
Biologics

1920 Dayton Avenue
PO Box 844
Ames, IA 50010

(515) 337-6100

Title: Proposals for Estimating Relative Potency
— Document Number: CVB-WI-0188.03
CONTROLLED//PROPIN//BASIC

Proposals for Estimating Relative Potency

Document Number: CVB-WI-0188 Revision: 03

Previous Number: PEL Reviewer Manual Ch. 4.6.2

Vault: CVB-Released

Section/Area: CVB-WI-PEL-REV

Effective Date: 25 Feb 2023

Notes:

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Signature Manifest Title: Proposals for Estimating Relative Potency Page 1 of 1
Release Date: 25 Feb 2023 Author: a Document Number: CVB-WI-0188.03
CONTROLLED//PROPIN//BASIC

Signature Manifest

Document Number: CVB-WI-0188 Revision: 03
Title: Proposals for Estimating Relative Potency
Effective Date: 25 Feb 2023

All dates and times are in Central Standard Time.

CVB-SOP-0102, CVB-WI-0188, CVB-WI-0286

Review for Doc Format

Name/Signature Title Date Meaning/Reason
TIANA BLANCO (TABLANCO) — Quality Management Assistant 23 Feb 2023, 11:38:52 AM Approved

Section Leader Final Approval

Name/Signature Title Date Meaning/Reason
CHRISTOPHER TONG (CTONG) CVB SR STAFF STATISTICIAN 23 Feb 2023, 12:26:53 PM Approved

Final Quality Check

Name/Signature Title Date Meaning/Reason
CROSLEY HERR (CRHERR) QA Specialist/CVB MC SME 25 Feb 2023, 04:40:37 PM Approved

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file:///E:/Mastercontrol/Pub/installedServices/publishingGL V T/adlib/work/mcjobs/all/330747F02.... 2/25/2023


Title: Proposals for oo Relative Potency
Release Date: 25 Feb 2023 Author: Document Number: CVB-WI-0188.03
CONTROLLED//PROPIN//BASIC

Proposals for Estimating Relative Potency

Relevant references and guidance documents:

e Veterinary Services Memorandum (VSM): 800.112, Appendix III, Section 2.2.9.
e Center for Veterinary Biologics (CVB) Notice:_13-18.
e CVB-SOP-0102, Using Software to Estimate Relative Potency

Introduction

CVB Notice 13-18 announced that CVB has discontinued use of RelPot software (described in
Supplemental Assay Method 318, now obsolete) as of Jan. 1, 2014. The CVB laboratory has
transitioned to using commercial software for estimating relative potency. The notice provides a
framework for firms to gradually transition to commercial software (or non-commercial
alternatives) for their outlines that currently include RelPot. CVB does not endorse any particular
alternate software, and equivalent calculations can be carried out in several available software
systems. What follows is purely for illustrative purposes. The discussion below applies both to
new assays and transition proposals for assays currently using RelPot/SAM 318.

Examples of relative potency calculations

e The CVB Statistics Section has provided examples for estimating relative potency using
SoftMax Pro (SMP) 6.2.1 (or later). These files are simplified versions of those used by
the CVB laboratory, and they serve to illustrate calculations that could readily be carried
out in several other software systems. The example files (“partiallylinear.sprx” and
“fulldose.sprx”) , formerly available at the Molecular Devices Community website,
which no longer exists, can now be found at https://github.com/ABS-dev/SMP

Examples for using GenS to calculate estimates of relative potency may be found on BioTek’s
website. Several files are available, allowing selection by curve fit type, horizontal axis units,
and competitive vs. immunometric. These files perform the same activity as in the above
example files and are another illustration of calculations that could be carried out in several
other software systems:

e Gen5 — Estimating Relative Potency sample files:
https://www.biotek.com/resources/technical-notes/estimating-relative-potency-for-cvb-

using-gen5-so ftware/

Firms may use the above links to access the examples. Firms (including those not using either of
the mentioned software) may consult CVB-SOP-0102, posted to the CVB website.

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Title: Proposals for Estimating Relative Potency
Release Date: 25 Feb 2023 Author: — Document Number: CVB-WI-0188.03
CONTROLLED//PROPIN//BASIC

This document describes the calculations implemented in the above files as applied in a general
way to situations where the procedure has not been fully specified in an outline. Ordinarily every
assay is expected to have a fully specified calculation procedure as outlined below.

Expectations for proposals for estimating relative potency

Proposals for estimating relative potency, either for transitioning assays that use RelPot, or for
new assays, should be routed to the Statistics Section for comment. For transitioning assays, a
full validation report is not expected. Rather, data from at least ten serial release tests may be

used to support the proposal. However, it is not acceptable for a firm to simply submit Outline
revisions without a transition proposal. For new assays, use of the proposed method should be
incorporated in the assay validation report that is normally expected per VSM 800.112.

Key items to look for include the following, and the Statistics Section will provide feedback with
respect to these points. The data model should be specified, e.g., parallel line analysis, or
nonlinear curves such as three-parameter logistic functions, with additional details specified
according to the table on the following page. The procedure should be fully specified, so that an
independent laboratory (such as CVB’s), possibly using different software, would be able to
reproduce the calculation. Revisions to the Special Outline or Outline of Production should
reflect the level of detail needed by an independent laboratory to carry out the procedure. Once a
transition is approved, the corresponding Special Outlines and/or Outlines of Production should
be updated promptly by the firm, and all mention of RelPot software and SAM 318 should be
expunged.

Please see the table on the next page for specific expectations.

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