In addition, the "drug tolerance level" is assigned based on the nominal amount of drug spiked into the validation sample, not the recovered amount that would be detected in the presence of surrogate, whereas in the study sample you'd be measuring the recovered amount of drug. It's very likely that any ADA is present in the study sample will be interfering with the PK assay measurement and probably causing under-recovery.
Original Message:
Sent: 06-13-2025 09:30
From: Eric Wakshull
Subject: Different Matrices for PK and Immunogenicity Assays
I agree with John's response. Unless your mAb binds to a component of the clot, which I've seen happen, then plasma and matrix should be the same for LBA. And drug tolerance is indeed a sliding scale, it uses a surrogate of usually high affinity and there is no simple "molar ratio" determination that can readily be applied across the various combinations you could run across in real samples.
Original Message:
Sent: 6/12/2025 11:41:00 AM
From: John Kamerud
Subject: RE: Different Matrices for PK and Immunogenicity Assays
Interesting question, Eric. I believe the choice of matrix often tends to be based on historical factors without a lot of scientific reasoning. For LBAs, the matrix of choice is serum more often than not, both for Pk and ADA. We should try to harmonize factors like this as much as possible.
But you asked about ongoing programs that are using different matrices. At least for LBAs and hybrid methods, my sense is that it is not likely to make a significant difference. (Of course, then we're left with deciding what constitutes a significant difference). But keep in mind that drug tolerance is not a solidly quantitative concept. 1) drug tolerance is determined using a surrogate positive control antibody and does not necessarily reflect the behavior of patient antibodies, and 2) drug tolerance changes depending on the concentration of antibody being detected, so it's a sort of "sliding scale'.
For these reasons I would not recommend doing any special testing to bridge the two matrices unless you have some scientific justification to think they may be different enough to impact the results of a non-quantitative ADA assay.
Original Message:
Sent: 6/11/2025 4:32:00 PM
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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