Examples of Types of PTM Modification Validations

PTM Method Modification Validations 

This document is meant to provide an overview of PTM method modification data  requirements based upon past method modifications submitted to the program. Each method  modification is assessed when submitted and the appropriate level determined based upon the  technology and function of the test method; therefore, data requirements for a method may  necessitate other data requirements not listed in this document to substantiate the method  modifications.  In an effort to maintain consistency in data requirements for PTM method modifications the  AOACRI has gathered details of all PTM method modifications from 2015‐2018 to assess the  data requirements imposed for each modification to provide a summary of data requirements  for modification categories.   PTM method modifications are reviewed by the AOACRI office and by the AOAC subject matter  expert, to determine if data are required to substantiate the method modifications. If data are  required, the AOAC Expert will detail the data requirements and assign a modification level.   Level 1 modifications are editorial changes to the package inserts, labels, method name  changes and method developer name changes. Data are not required to substantiate these  modifications; however, an updated license agreement is required for method name and  method developer name changes.  If package insert revisions are substantial within the method procedure the revised insert is  reviewed by an AOAC subject matter expert to assess whether an evaluation of the method  workflow is needed. Internal data may be requested to determine that not modification  validation data are needed.  Level 2 modifications either impact the method claim or improve the method function. Claim  changes include addition of claimed matrices, addition of a reference method and/or expanded  inclusivity. Kit changes include a wide variety of modifications. Internal data are often reviewed  before specific modification data requirements are determined There are three PTM modification levels:   Level 1 ‐ Editorial changes   Level 2 ‐ Minor method modifications.   Level 3 ‐ Major method modifications. 

Table 1. Level 2 Method Modification Examples and Data Requirements  Method Modification  Data Requirement 

Matrix extensions up to four matrices,  depending on the number of matrices  included in the original validation claim.  For example:  Original claim 4 – level 2 modification  would be acceptable for only one matrix.  More than five claimed matrices require  an independent lab study.  Original claim 6 – level 2 modification for  the addition of 4 more matrices is  acceptable without an independent study.   Changes to non‐critical reagents  (lyophilization, Taq,), does not include  reagents that may affect  specificity/sensitivity such as new  antibodies or primer/probe sequences.  Enhancement of method workflow (non‐ critical steps)  Non‐reagent changes, such as changes to  the cartridge or increase in number of  wells  Changes in matrix sample sizes 

Matrix comparison study for all matrices. 

Matrix comparison study for all matrices and samples  sizes with the reference method samples size. 

Inclusivity/exclusivity and matrix comparison data for a  subset of claimed matrices. In some instances lot‐to‐lot,  stability and robustness required. 

Testing of 3 matrices inoculated post enrichment;  inclusivity/exclusivity; lot‐to‐lot studies.  

Dilution to extinction experiment. Matrix study 1‐3  matrices tested depending upon the overall matrix claim  for the method (matrices chosen by AOAC).  Inclusivity/exclusivity and matrix comparison data for a  subset of claimed matrices (matrices chosen by AOAC).  Where possible, the testing of frozen lysates from  previous validation on new instrument.  Inclusivity/exclusivity and matrix comparison data for a  subset of claimed matrices.  

New instrument, thermal cycler, PCR  platform or other 

Addition of DNA preparation methods 

Addition or update reference method 

Matrix comparison study for a subset of matrices. 

Expansion of the inclusivity claim within  the same target analyte species; for  example, in the case of expansion of  Listeria species claim.  Expansion of stability claim for reagents/

Inclusivity and exclusivity testing (blind‐randomized). 

whole kit  

Stability study of frozen and refrigerated product; growth  curve analysis, shelf‐life data and lot‐to‐lot; real‐time  stability data; testing on several instruments.   Depending upon the change and the technology, specific  details will need to be reviewed by the AOAC volunteer  expert.  Lot‐to‐lot comparison testing of 1 lots from previous  manufacturing facility and 3 lots from the new  manufacturing facility. One target strain at 10XLOD  tested using 10 replicate samples with fractional recovery  results.  Comparison of QA/QC data has been used when old lots  are no longer available. 

Software changes 

Manufacturing location changes 

Changes in raw agar ingredients 

Inclusivity/exclusivity 

Table 1 details a summary of level 2 method modifications and the data requirements to  substantiate the changes. For most level 2 modifications a matrix comparison study is required,  some inclusivity/exclusivity testing and possible robustness testing. For manufacturing location  changes a lot‐to‐lot comparison testing scheme is used with lots from the previous  manufacturing site to those lots from the current site. However, in instances where there are  no available kits produced at the previous manufacturing site AOACRI has accepted QA/QC  comparison data from kits produced at the new manufacturing site compared to QA/QC data  from the previous manufacturing site.  Level 3 modifications are major changes to the test method, components and/or workflow.  Internal and independent study data are required to substantiate the method modification  changes. 

Table 2. Level 3 Method Modification Examples and Data Requirements  Method Modification  Data Requirement 

Inclusivity/exclusivity, matrix comparison for subset of  claimed matrices, robustness testing and an  independent laboratory study. 

Changes in enrichment media, extraction  buffer, temperature and/or times.  Matrix extensions where the number of  matrices being added is exceeds the  independent laboratory testing threshold  of 1 matrix independently tested for every  5 claimed matrices in the original  validation claim.  

Matrix comparisons for all matrices being added and an  independent laboratory study for 1 matrix for every 5  matrices in the total claim.

For example:  Original claim 3 matrices – 6 matrices will  be added bringing the total number of  matrices to 9, triggering an independent  laboratory study of two matrices (one  matrix previously tested and one new  matrix tested at the independent  laboratory).   Original claim 1 matrix – 5 matrices will be  added bringing the total number of  matrices to 6, triggering an independent  laboratory study of two matrices (one  matrix previously tested and one new  matrix tested at the independent  laboratory).   Reagent formulation of the amplification  reagent master mix  Addition or removal of a test step (for  example‐LFD step, second PCR step,  confirmation step or molecular verification  step) 

Inclusivity/Exclusivity, matrix comparison for subset of  claimed matrices and an independent laboratory study.  

Inclusivity/Exclusivity, matrix comparison for subset of  claimed matrices, and independent laboratory study. 

Table 2 details a summary of level 3 method modifications and the data requirements to  support the changes. Major modifications require substantial evaluation to ensure the modified  method still meets the method performance claims. Examples of major method changes are  changes to the enrichment protocol, addition of more than five matrices, change to a major kit  component, reformulation of master mix reagents and addition or removal of a test step. 

Reference Methods and Modifications 

Method modifications will utilize the most current reference method available regardless of the  reference method version used in the original validation study. 

Publication of Method Modifications

Currently,  Journal of AOAC INTERNATIONAL  (JAOAC) publication of method modification study  reports is not required. Publication of a method modification study report is at the discretion of  the method developer.