Bioequivalence and Bioavailability Forum • Little thing which changes almost everything in EEU

Beholder
★

Russia,
2018-10-18 11:08
(2188 d 23:52 ago)

Posting: # 19464
Views: 5,546

Little thing which changes almost everything in EEU [Bioanalytics]

Hi to everybody!

Recently (IMHO, too late for me), I have revealed that EEU BEQ GL has interesting note in validation chapter which is not presented in EU BEQ GL, namely:
“Lower limit of quantification
14. The lower limit of quantification (LLOQ) is the lowest concentration of analyte in a sample that can be reliably quantified with acceptable accuracy and precision. The LLOQ is considered to be the smallest standard calibration sample (as indicated in the “Accuracy” and “Precision” subsections of this section). In this case, the signal of the analyte from the sample with the LLOQ must be not less than 5 times greater than the value of the blank sample. The LLOQ cannot be higher than 5% of C_max (the minimum C_max value from the entire sample of subjects) ).”

EU GL states the same but without blue text.
So, IMHO:

it is quite “butthurting” since we need to set LLOQ before analyzing biosamples, but EEU BEQ GL asks to set LLOQ according to minimal C_max from the entire sample of subjects but not the average C_max. It may lead us to following steps:
1. set the C_max in advance
2. analyze biosamples
3. if you are not lucky and your LLOQ higher than 5% of minimal C_max of any subject in the study, what is quite possible because of high variability of C_max, revalidate your LLOQ with lower value
4. analyze biosamples again
it is also quite possible that there will be no opportunity to lower the LLOQ due to limitation of the LC/MSMS instrument.

What do you do in case when LLOQ higher than 5% mean C_max? I mean in EU countries.
What is your opinion, where it could came from? Any old EU BEQ rule?

Im quite surprised that the sentence about minimal C_max was not excluded during public consultations.

Edit: EEU GL linked. [Helmut]

—
Best regards
Beholder

Helmut
★★★

Vienna, Austria,
2018-10-18 14:28
(2188 d 20:32 ago)

@ Beholder
Posting: # 19468
Views: 5,013

LLOQ ≤5% of expected or observed Cmax?

Post reply

Hi Beholder,

first of all congratulations for mastering the forum’s tricky syntax of nested lists. ;-)

❝ […] I have revealed that EEU BEQ GL has interesting note in validation chapter which is not presented in EU BEQ GL, namely:

❝ “[…] The LLOQ cannot be higher than 5% of C_max (the minimum C_max value from the entire sample of subjects) ).”

AFAIK, the Russian language leaves a lot of space for interpretation. Is the common meaning of »не должен« really equivalent to “cannot” or “must not”? Since I don’t speak Russian, I have to rely on A(un)I: , Yandex, and Bing translate it as “should not” – which is exactly the phrase used in the EMA’s GL.

If it is mandatory (your #1), very stupid. #2 is a show-stopper.

I can only tell you what is done in studies for the EMA. Use an estimate of the lowest expected C_max and base the LLOQ on it. Depending on the expected variability CROs commonly opt for 2% or even 1% of the average C_max. Nerds like me work with modeling, stuff like that (replace t_½ with C_max), or simulations. Of course, if individual data are available (pilot or other studies), better. Never saw revalidation. If you are cautious, exclude the subject. If that doesn’t occur too often, the loss in power is small.

—
Dif-tor heh smusma 🖖🏼 Довге життя Україна!
Helmut Schütz

The quality of responses received is directly proportional to the quality of the question asked. 🚮
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Beholder
★

Russia,
2018-10-18 15:59
(2188 d 19:01 ago)

(edited on 2018-10-19 08:34)
@ Helmut
Posting: # 19469
Views: 4,911

LLOQ ≤5% of average or minimal Cmax?

Post reply

Hi Helmut,

❝ first of all congratulations for mastering the forum’s tricky syntax of nested lists. ;-)

THX. I reached it finally. Years passed. :-D

❝ AFAIK, the Russian language leaves a lot of space for interpretation. Is the common meaning of »не должен« really equivalent to “cannot” or “must not”? Since I don’t speak Russian, I have to rely on A(un)I: , Yandex, and Bing translate it as “should not” – which is exactly the phrase used in the EMA’s GL.

You are right regarding the meaning. Yes, it is rather "should not". But looks like I was not clear enough. I was talking about text which is marked with blue: "...(the minimum Cmax value from the entire sample of subjects)". It is one story when we take 5% from average C_max, but it is another story when we take 5% from minimum value of C_max in volunteers cohort. My question was caused by an article where such information was presented: "According to the literature, the minimum arithmetic average Cmax is 1,479.61 ng / ml (when taking atazanavir at a dose of 300 mg) [15]. Thus, the LLOQ should not exceed 74 ng / ml. However, it should be taken into account that, in accordance with the requirements of the EAEU, the LLOQ is calculated based on the minimum value of Cmax from the entire sample of subjects [4]. In the analyzed studies, the minimum value of Cmax was 60.8 ng / ml, while the calculated value of LLOQ is 3 ng / ml."

Edited: link to the article updated.

—
Best regards
Beholder

Helmut
★★★

Vienna, Austria,
2018-10-18 20:20
(2188 d 14:41 ago)

@ Beholder
Posting: # 19472
Views: 4,901

LLOQ ≤5% of individual Cmax-values

Post reply

Hi Beholder,

❝ You are right regarding the meaning. Yes, it is rather "should not". But looks like I was not clear enough.

Oh, you were.

❝ My question was caused by an article […]

The link is not working. Can you check?

❝ According to the literature, the minimum arithmetic average Cmax is 1,479.61 ng / ml.

Arithmetic means of concentrations are stupid. Reporting concentrations to six significant digits even more. I assume that the “minimum arithmetic average C_max” was calculated from different studies; each with a dose of 300 mg.

❝ Thus, the LLOQ should not exceed 74 ng / ml.

OK, 5% of a doubtful value (likely the geometric mean is lower). Setting the LLOQ based on (any) average is stupid as well.

❝ However, it should be taken into account that, in accordance with the requirements of the EAEU, the LLOQ is calculated based on the minimum value of Cmax from the entire sample of subjects.

Makes sense. (Lack of) carry-over has to be assessed on a subject-level. See what I wrote at the end of my previous post. We cannot get an unbiased treatment-effect in the presence of true carryover. Hence, we have to avoid CO by design. One method is to assess pre-dose concentrations in periods >1. And yes, make sure that we are able to measure 5% of every subject’s C_max after each treatment.

❝ In the analyzed studies, the minimum value of Cmax was 60.8 ng / ml,

Wow, that’s extreme! Not clear to me whether the authors are referring to the same dose of 300 mg in all studies.

❝ while the calculated value of LLOQ is 3 ng / ml."

5% again. If the same dose was used in all studies (I doubt it) it would be a nightmare:

LLOQ 3 ng/mL makes sense.
Given such a high between-subject variability, what will be the upper limit of individual C_max-values? 3,000 ng/mL, 5,000 ng/mL, or even higher? Would drive most detectors to the edge.
Yes, one could use a quadratic calibration but that would decrease the accuracy at high concentrations (the curve is getting flatter). Set the ULOQ not that high, but dilute and reanalyze samples >ULOQ?

PS: It can be even worse. Sometimes in studies in patients (mainly phase III) different LLOQs are used. Might be caused by limited sample volumes or interactions with co-medications. The world is not ideal.

—
Dif-tor heh smusma 🖖🏼 Довге життя Україна!
Helmut Schütz

The quality of responses received is directly proportional to the quality of the question asked. 🚮
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mittyri
★★

Russia,
2018-10-18 21:50
(2188 d 13:11 ago)

@ Helmut
Posting: # 19473
Views: 4,763

LLOQ ≤5% of individual Cmax-values

Post reply

Hi Helmut and Beholder,

Let's take a hypothetical example
Due to high intersubject variability we got some periods for some (not many) subjects less than 5% of LLOQ.
1. Would you ask analytical lab to revalidate the method and reanalyze those profiles again or since analytical part is over, there's no way to change the method when playing part starts (Cmax estimation is PK part, right?)

2. Do you think the reasons to exclude that subjects are feasible enough?
Have you ever been asked to do that?

For me this is mind-blowing since the applicants should invest money not only in dropouts, but in extreme metabolizes too! The power loss for replicate designs is more significant (double the subject data)

PS: moreover, that criterion (Cmax< 20*LLOQ) is not listed in EMA GL as a reason for subject exclusion

—
Kind regards,
Mittyri

Beholder
★

Russia,
2018-10-19 11:43
(2187 d 23:18 ago)

@ mittyri
Posting: # 19476
Views: 4,745

An outlier?

Post reply

Hi Helmut and mittyri!

❝ The link is not working. Can you check?

Please check now. Should work. THX to Moderator.

❝ Reporting concentrations to six significant digits even more. I assume that the “minimum arithmetic average Cmax” was calculated from different studies; each with a dose of 300 mg.

Looks like the value 1,479.61 ng / ml was taken from PAR of one big Indian pharmaceutical company. I’m not sure that it is average of different studies. BTW, what about seven and even eight significant digits (see PAR, pg 20)? :-D

❝ In the analyzed studies, the minimum value of Cmax was 60.8 ng / ml,

IMHO, that is actually the case. According to the article (Table 1), only 300 mg and 400 mg doses (not strengths) were included to the analysis. So looks like 60.8 ng / ml is an outlier due to extreme fast metabolizing?

—
Best regards
Beholder