Bioanalytical Community

Greetings! I would like to ask how do community members define Drug Tolerance (DT) for ADA methods. For example, in order to judge a sample that tested Negative in the screening assay as ADA-Negative, the concentration of the drug present in the sample should be at or below the method's DT level. Is everyone still using DT value corresponding to 100 ng/mL of Positive Control for this purpose, or do you use DT value corresponding to the "true" LPC concentration that was statistically calculated based on the method's sensitivity? Thank you!

Hi Anna, If we go by the definition, "the highest concentration of free drug that allows for the detection of ADA at the assay sensitivity levels," then the target DT is the Cmax at the LPC level. By this measure, DT will assess that the assay sensitivity, as measured by the low positive control, does not change or is unaffected when the drug is present at the maximal possible available concentration. In other words, the assay is sensitive enough to detect ADA at all possible PK levels.

Therefore, we typically test the PC at least at 0 PC, LPC, 100 ng/mL (the magic number, which may not be relevant to a given assay), and HPC. The bioA report includes the drug level determined at least at 0, LPC, and 100 ng/ml. In my personal opinion, 100 ng/ml is an artificially employed parameter that serves as a guide and has no bearing on the assay itself.

Greetings! I would like to ask how do community members define Drug Tolerance (DT) for ADA methods. For example, in order to judge a sample that tested Negative in the screening assay as ADA-Negative, the concentration of the drug present in the sample should be at or below the method's DT level. Is everyone still using DT value corresponding to 100 ng/mL of Positive Control for this purpose, or do you use DT value corresponding to the "true" LPC concentration that was statistically calculated based on the method's sensitivity? Thank you!

Greetings! I would like to ask how do community members define Drug Tolerance (DT) for ADA methods. For example, in order to judge a sample that tested Negative in the screening assay as ADA-Negative, the concentration of the drug present in the sample should be at or below the method's DT level. Is everyone still using DT value corresponding to 100 ng/mL of Positive Control for this purpose, or do you use DT value corresponding to the "true" LPC concentration that was statistically calculated based on the method's sensitivity? Thank you!

Greetings! I would like to ask how do community members define Drug Tolerance (DT) for ADA methods. For example, in order to judge a sample that tested Negative in the screening assay as ADA-Negative, the concentration of the drug present in the sample should be at or below the method's DT level. Is everyone still using DT value corresponding to 100 ng/mL of Positive Control for this purpose, or do you use DT value corresponding to the "true" LPC concentration that was statistically calculated based on the method's sensitivity? Thank you!

To define drug tolerance, I believe most people following the recommendation (or a variation) of the 'Report on the AAPS Immunogenicity Guidance Forum' (https://doi.org/10.1208/s12248-019-0328-8) which states: Concentrations of drug selected for evaluation should target concentrations that are at lower-than-expected, at expected, and at higher-than-expected levels for sample trough. PC should be tested at the LPC concentrations, 100, 250, and 500 ng/mL, and potentially higher concentrations. Drug tolerance should be evaluated in both the screening and confirmatory tiers.

Of the concentration tested, the 100, 250 and 500 ng/mL of PC are the most relevant for defining drug tolerance. Drug tolerance at the LPC is pretty irrelevant - the LPC is typically selected to be very close to the screening cut-point to monitor/control that there is consistent sensitivity between different runs. By this selection process, the LPC is often intolerant to even low drug levels.

Even though drug tolerance is defined relative to a 'surrogate' positive control, if the PC is a rabbit polyclonal, then I think we have enough experience in the field to say that if you can detect a positive response from 100 or 250 ng/mL PC in the presence of Ctrough levels of drug, then you're very likely to detect any ADA that are clinically relevant.

Best wishes,

Rob

Greetings! I would like to ask how do community members define Drug Tolerance (DT) for ADA methods. For example, in order to judge a sample that tested Negative in the screening assay as ADA-Negative, the concentration of the drug present in the sample should be at or below the method's DT level. Is everyone still using DT value corresponding to 100 ng/mL of Positive Control for this purpose, or do you use DT value corresponding to the "true" LPC concentration that was statistically calculated based on the method's sensitivity? Thank you!

To define drug tolerance, I believe most people following the recommendation (or a variation) of the 'Report on the AAPS Immunogenicity Guidance Forum' (https://doi.org/10.1208/s12248-019-0328-8) which states: Concentrations of drug selected for evaluation should target concentrations that are at lower-than-expected, at expected, and at higher-than-expected levels for sample trough. PC should be tested at the LPC concentrations, 100, 250, and 500 ng/mL, and potentially higher concentrations. Drug tolerance should be evaluated in both the screening and confirmatory tiers.

Of the concentration tested, the 100, 250 and 500 ng/mL of PC are the most relevant for defining drug tolerance. Drug tolerance at the LPC is pretty irrelevant - the LPC is typically selected to be very close to the screening cut-point to monitor/control that there is consistent sensitivity between different runs. By this selection process, the LPC is often intolerant to even low drug levels.

Even though drug tolerance is defined relative to a 'surrogate' positive control, if the PC is a rabbit polyclonal, then I think we have enough experience in the field to say that if you can detect a positive response from 100 or 250 ng/mL PC in the presence of Ctrough levels of drug, then you're very likely to detect any ADA that are clinically relevant.

Best wishes,

Rob

Greetings! I would like to ask how do community members define Drug Tolerance (DT) for ADA methods. For example, in order to judge a sample that tested Negative in the screening assay as ADA-Negative, the concentration of the drug present in the sample should be at or below the method's DT level. Is everyone still using DT value corresponding to 100 ng/mL of Positive Control for this purpose, or do you use DT value corresponding to the "true" LPC concentration that was statistically calculated based on the method's sensitivity? Thank you!

Greetings! I would like to ask how do community members define Drug Tolerance (DT) for ADA methods. For example, in order to judge a sample that tested Negative in the screening assay as ADA-Negative, the concentration of the drug present in the sample should be at or below the method's DT level. Is everyone still using DT value corresponding to 100 ng/mL of Positive Control for this purpose, or do you use DT value corresponding to the "true" LPC concentration that was statistically calculated based on the method's sensitivity? Thank you!

Hi Anna,

                Let me add my two pennies. I understand that in case of drug concentration in the sample exceeding drug tolerance, you are going to report the negative result as "inconclusive". I really don't like the "inconclusive" category. First of all, "inconclusive" suggests that the assay is unable to generate any "conclusive" results. However, when we have a positive result in the presence of drug concentration > drug tolerance, we don't flag this result as somehow untrustworthy. Second, when a high drug concentration is present in the sample, a negative result is very conclusive. It means that ADA level in this sample is lower than that established during drug tolerance experiments. For example, if your assay can detect 200 ng/mL of positive control in the presence of 20 μg/mL of drug, then a negative result for a sample with 20 μg/mL of drug on board means ADA level is <200 ng/mL. The same reasoning could be applied to all ADA-negative samples, regardless of the drug concentration. This is because our assays have limited sensitivity, so "negative" really means "below the limit of detection of the positive control antibody evaluated in the assay used for sample testing". 

I think this illustrates a larger conceptual problem with reporting immunogenicity results. We mix categorical nominal values (e.g. positive, negative, cat, dog) with categorical ordinal values (e.g. low positive, high positive, medium spicy) with quantitative values based on a positive control antibody (e.g. in 99% of tests 10 ng/mL of the positive control gives a response that is equal to or higher than statistically determined cut point for this assay). Methinks, immunogenicity testing is ripe for a general revision, and we should reconsider it based on 20 years of data generated since the first white papers on the subject.

Best regards,

Robert

Greetings! I would like to ask how do community members define Drug Tolerance (DT) for ADA methods. For example, in order to judge a sample that tested Negative in the screening assay as ADA-Negative, the concentration of the drug present in the sample should be at or below the method's DT level. Is everyone still using DT value corresponding to 100 ng/mL of Positive Control for this purpose, or do you use DT value corresponding to the "true" LPC concentration that was statistically calculated based on the method's sensitivity? Thank you!