Dear Eric,
The main historical driver for using serum instead of plasma was because the matrix was "cleaner", in that the coagulation pulled a lot of "gunk" out of the matrix that generated more nonspecific background. So the biggest difference in the two assays is typically sensitivity.
Unless the drug target is in the clot, however, we generally see the same results in serum or plasma for PK and ADA once in the range of the assays. We have honestly evolved to the point where you might be able to do the ADA in plasma if you really wanted to since our assays are also getting crazy good. An exception might be cell-based neutralizing antibody assays where the anticoagulant can mess with the cells, or diluted samples might start clotting as the anticoagulant is diluted as well.
We have honestly evolved to the point where you might be able to do the ADA in plasma if you really wanted to since we can do in whole blood as well, but I also don't see huge issues in using plasma and serum even if the target does partition to the clot and the concentration in serum would be a little higher. Drug tolerance is a sliding scale, not all or nothing, so you wouldn't be turning it off simply by switching to serum for ADA. And evaluating impact to PK from ADA seroconversion is a relative assessment, not absolute. So unless the difference is not linear, you would come to the same conclusion either way.
This is my initial response, and I'm sure there are other cases where it is a problem. But generally I think it is okay.
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Joleen White Ph.D.
AAPS 2024 Global Health Community Chair
Bioanalytical 101 Course Development
Senior Advisor
BioData Solutions LLC
[email protected]
Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
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Original Message:
Sent: 06-11-2025 16:31
From: Eric Woolf
Subject: Different Matrices for PK and Immunogenicity Assays
The use of LC/MS for the quantitation of "large molecules" such as antibodies, proteins, peptides, and antibody drug conjugates is gaining popularity. The typical matrix for such assays is plasma. Assessment of immunogenicity is generally required during clinical development for these modalities. Anti-drug antibody and neutralizing antibody assays are historically ligand-binding based and utilize serum as a matrix.
As part of the validation of the immunogenicity assays, the drug tolerance of the assay needs to be determined. The results of the quantitative assays being used for pharmacokinetic assessment are then compared with the drug tolerance of immunogenicity assays to ensure that the immunogenicity results of the collected samples are not influenced by the presence of drug.
Is it a concern if the drug level assay and immunogenicity assays are run in different matrices (i.e. plasma for PK and serum for immunogenicity)?
For small molecules, there are documented cases where serum levels are different from plasma levels. These may be due, for example, to differences in stability between the matrices or cation binding of analytes impacting distribution into red blood cells, which may be disturbed when EDTA is used as the anticoagulant.
Are there similar issues with "large molecules"? While using the same matrix for both quantitative and immunogenicity assays is the "ideal" solution, in the case of ongoing programs where the two different matrices are being used, what assessments are recommended to bridge the plasma results to serum to ensure that immunogenicity results are not assessed against an inaccurate drug concentration in the sample?
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Eric Woolf, Ph.D., FAAPS
Souderton, PA
Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
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