Parallelism and Matrix Interference: The Importance of Nomenclature (Part 1)

For the bioanalytical validation of PK and biomarker assays, certain terms can have multiple definitions depending on the assay.  In this two part blog series we will discuss the importance of nomenclature and discuss some commonly confused or misunderstood terms.

As an example, the term parallelism is used for both PK and biomarker assays, but the meaning and testing implications are different.  For biomarker assays, parallelism is performed on commercially available samples during development and pre-study validation to show that endogenous biomarkers behave in similar fashion to the calibrator of the assay.  Contrarily, for PK assays, parallelism is not routinely performed and only used as an investigational tool. Parallelism is performed in study samples (pooled or individual) to determine if there is a specific issue with the sample of interest (ex. presence of ADA or aggregation).

Another example is the term “Matrices interference” and its use in PK and immunogenicity.  During the validation of a PK assay, a minimum of 6 normal individual samples are run at the minimum required dilution (MRD) to determine if any of the naïve samples read above the lower limit of quantitation (LLOQ). The expectation is that the validated assay will not detect any drug in naïve samples unless the drug is an endogenous molecule as presented in the 2013 draft FDA guidance:

“Evidence should be provided that the substance quantified is the intended analyte. Analyses of blank samples of the appropriate biological matrix (plasma, urine, or other matrix) should be obtained from at least six sources. Each blank sample should be tested for interference, and selectivity should be ensured at the lower limit of quantification (LLOQ).  Potential interfering substances in a biological matrix include endogenous matrix components; metabolites; decomposition products; and, in the actual study, concomitant medication and other xenobiotics. If the method is intended to quantify more than one analyte, each analyte should be tested to ensure that there is no interference”.

Whereas during immunogenicity validation, the 2009 draft FDA guidance describes matrix interference as the following:

“Matrix effect: The direct or indirect alteration or interference in response due to the presence of unintended analytes (for analysis) or other interfering substances in the sample.”

To determine Matrix effect, the FDA guidance recommends testing any components in the matrix other than antibodies that can interfere with assay results.  The greatest concern is the product’s presence in the matrix, or its endogenous counterpart.  Specifically, if large quantities of product-related material are present in sera/plasma, that material can prevent detection of antibodies in the test format. The FDA recommends that the applicant addresses such possibilities early (preclinical and phase I or early phase II). The applicant should also examine this issue by deliberately adding known amounts of purified antibodies into assay buffer in the absence or presence of different quantities of the protein under consideration.

Parallelism and matrix interference are just two examples out of a vast list of homonyms in the Bioanalytical world.  It is very important for the sponsor and the CRO to understand the definition of each depending on the assay types.  Stay tuned for our next blog that will list the terms and definitions for each type of assay.

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