Apologies for lack of clarity. I meant raise the LLOQ to the lowest concentration that hemolysis interference does not fail. But I agree the way I actually wrote it is potentially interesting as well. I know that I have submitted data that had different LLOQ either due to insufficient sample to reanalyze at lower dilution or insufficient sample to reanalyze after having to raise the LLOQ due to a calibrator failure. But that was very small numbers in a data set.
I think the key element would be following a consistent process, so you would have to build in checking hemolysis status to demonstrate that there was no bias in whether or not a sample was considered hemolyzed. Potentially even grouping all hemolyzed samples together so that you can apply a run-specific LLOQ. The downstream impact would need to be discussed with the PK scientist as LLOQ results can be treated as zero or half LLOQ depending on the analysis plan. Either way, it would be important to understand how using different LLOQ values for hemolyzed samples would impact the analysis.
Being an advocate of patient-centric sampling, I am going a completely different direction with my thoughts from the previous suggestion. What if you just used whole blood to do the equivalent of "spiking" interfering substance.
That said, I hope some people have some suggestions on assay modifications to disrupt the interference. @Fumin Li, potentially a topic for a future Chromatographic Analysis Working Group?
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Joleen White Ph.D.
AAPS 2024 Global Health Community Chair
Bioanalytical 101 Course Development
Senior Bioassay Development Lead
Gates Medical Research Institute
Cambridge MA
[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: 06-07-2024 12:03
From: Shashank Gorityala
Subject: Hemolysis assessment in mAb therapeutic PK by Direct Digestion-based bottom up LC/MS
Hello Joleen,
Thank you for your response. Unfortunately, we do not have access to a stable isotope-labeled protein version as an internal standard. It would be worthwhile to evaluate whether the drug remains consistent when incubated in the hemolyzed matrix (1FT cycle) in case there are equilibrium dynamics that need to be established before analysis.
In my experience, predicting the number of hemolyzed samples in a given set can be challenging. It often depends on the collection site and the phlebotomist. Considering raising the lower limit of quantification (LLOQ) for hemolyzed samples is an interesting idea. Have you previously explored this route without encountering any regulatory risks?
Thanks!
Shashank
<cib-overlay> </cib-overlay>
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Shashank Gorityala
Associate Director, LC/MS Bioanalysis
BioAgilytix
Durham NC
[email protected]
Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
Original Message:
Sent: 06-06-2024 13:00
From: Joleen White
Subject: Hemolysis assessment in mAb therapeutic PK by Direct Digestion-based bottom up LC/MS
While not the most affordable response, switching to a stable label protein as your internal standard rather than the peptide could likely solve the problem. That's a huge advantage of the chromatographic techniques if you can fully match the sample. Of course you'd need to let the spiked internal standard equilibrate with the sample for a while before treatment.
Another solution that I saw from a colleague for a different type of interference was spiking the interfering substance to equilibrium in all samples and then just letting that be a baseline. Might be difficult for hemolysis because there is no guarantee that the interference is a single component.
A more operational solution that may be feasible depending on how many hemolyzed samples and how many samples are expected to be impacted is to simply raise the LLOQ for hemolyzed samples to the lowest concentration that does pass interference. Or just not accept hemolyzed samples, although the latter is practically impossible in oncology and pediatric trials.
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Joleen White Ph.D.
AAPS 2024 Global Health Community Chair
Bioanalytical 101 Course Development
Senior Bioassay Development Lead
Gates Medical Research Institute
Cambridge MA
[email protected]
Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
Original Message:
Sent: 06-06-2024 11:51
From: Shashank Gorityala
Subject: Hemolysis assessment in mAb therapeutic PK by Direct Digestion-based bottom up LC/MS
Dear BA community,
I am seeking feedback regarding an issue we've encountered. Thank you in advance for your insights.
We are experiencing concentration-dependent hemolysis assessment failure for a monoclonal antibody (mAb) drug in human matrix. This issue arises during direct digestion-based signature peptide (CDR peptide) quantification using an LC-QqQ-MS workflow. Notably, the problem is isolated and consistent with the low-quality control (LQC-Hemolysis) level trending low, while the high-quality control (HQC-Hemolysis) meets criteria. We use an isotope-labeled peptide as an internal standard. Despite optimizing denaturation, alkylation, reduction, and digestion conditions, we suspect that hemolyzed matrix components may be binding the target mAb, thereby reducing digestion efficiency compared to non-hemolyzed matrix. Have any effective strategies been successful in resolving this/similar kind of issue?
Thank you!
Shashank
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Shashank Gorityala
Associate Director, LC/MS Bioanalysis
BioAgilytix
Durham NC
[email protected]
Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
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