4.1 Key Reagents 619
4.1.1 Reference Standard 620
The reference standard should be well characterised and documented (e.g., CoA and origin). A 621
biological drug has a highly complex structure and its reactivity with binding reagents for 622
bioanalysis may be influenced by a change in the manufacturing process of the drug substance.
623
It is recommended that the manufacturing batch of the reference standard used for the 624
preparation of calibration standards and QCs is derived from the same batch of drug substance 625
as that used for dosing in the nonclinical and clinical studies whenever possible. If the reference 626
standard batch used for bioanalysis is changed, bioanalytical evaluation should be carried out 627
prior to use to ensure that the performance characteristics of the method are within the 628
acceptance criteria.
629
4.1.2 Critical Reagents 630
Critical reagents, including binding reagents (e.g., binding proteins, aptamers, antibodies or 631
conjugated antibodies) and those containing enzymatic moieties, have direct impact on the 632
results of the assay and, therefore, their quality should be assured. Critical reagents bind the 633
analyte and, upon interaction, lead to an instrument signal corresponding to the analyte 634
concentration. The critical reagents should be identified and defined in the assay method.
635
Reliable procurement of critical reagents, whether manufactured in-house or purchased 636
commercially, should be considered early in method development. The data sheet for the critical 637
reagent should include at a minimum identity, source, batch/lot number, purity (if applicable), 638
concentration (if applicable) and stability/storage conditions (Refer to Table 1). Additional 639
characteristics may be warranted.
640
A critical reagent lifecycle management procedure is necessary to ensure consistency between 641
the original and new batches of critical reagents. Reagent performance should be evaluated 642
using the bioanalytical assay. Minor changes to critical reagents would not be expected to 643
influence the assay performance, whereas major changes may significantly impact the 644
performance. If the change is minor (e.g., the source of one reagent is changed), a single 645
comparative accuracy and precision assessment is sufficient for characterisation. If the change 646
is major, then additional validation experiments are necessary. Ideally, assessment of changes 647
will compare the assay with the new reagents to the assay with the old reagents directly. Major 648
changes include, but are not limited to, change in production method of antibodies, additional 649
blood collection from animals for polyclonal antibodies and new clones or new supplier for 650
monoclonal antibody production.
651
Retest dates and validation parameters should be documented in order to support the extension 652
or replacement of the critical reagent. Stability testing of the reagents should be based upon the 653
performance in the bioanalytical assay and be based upon general guidance for reagent storage 654
conditions and can be extended beyond the expiry date from the supplier. The performance 655
parameters should be documented in order to support the extension or replacement of the critical 656
reagent.
657
4.2 Validation 658
When using LBA, study samples can be analysed using an assay format of 1 or more well(s) 659
per sample. The assay format should be specified in the protocol, study plan or SOP. If method 660
development and assay validation are performed using 1 or more well(s) per sample, then study 661
sample analysis should also be performed using 1 or more well(s) per sample, respectively. If 662
multiple wells per sample are used, the reportable sample concentration value should be 663
determined either by calculating the mean of the responses from the replicate wells or by 664
averaging the concentrations calculated from each response. Data evaluation should be 665
performed on reportable concentration values.
666
4.2.1 Specificity 667
Specificity is evaluated by spiking blank matrix samples with related molecules at the maximal 668
concentration(s) of the structurally related molecule anticipated in study samples.
669
The accuracy of the target analyte at the LLOQ and at the ULOQ should be investigated in the 670
presence of related molecules at the maximal concentration(s) anticipated in study samples. The 671
response of blank samples spiked with related molecules should be below the LLOQ. The 672
accuracy of the target analyte in presence of related molecules should be within ±25% of the 673
nominal values.
674
In the event of non-specificity, the impact on the method should be evaluated by spiking 675
increasing concentrations of interfering molecules in blank matrix and measuring the accuracy 676
of the target analyte at the LLOQ and ULOQ. It is essential to determine the minimum 677
concentration of the related molecule where interference occurs. Appropriate mitigation during 678
sample analysis should be employed, e.g., it may be necessary to adjust the LLOQ/ULOQ 679
accordingly or consider a new method.
680
During method development and early assay validation, these “related molecules” are 681
frequently not available. Additional evaluation of specificity may be conducted after the 682
original validation is completed.
683
4.2.2 Selectivity 684
Selectivity is the ability of the method to detect and differentiate the analyte of interest in the 685
presence of other “unrelated compounds” (non-specific interference) in the sample matrix. The 686
matrix can contain non-specific matrix component such as degrading enzymes, heterophilic 687
antibodies or rheumatoid factor which may interfere with the analyte of interest.
688
Selectivity should be evaluated at the low end of an assay where problems occur in most cases, 689
but it is recommended that selectivity is also evaluated at higher analyte concentrations.
690
Therefore, selectivity is evaluated using blank samples obtained from at least 10 individual 691
sources and by spiking the individual blank matrices at the LLOQ and at the high QC level.
692
The response of the blank samples should be below the LLOQ in at least 80% of the individual 693
sources.
694
The accuracy should be within ±25% at the LLOQ and within ±20% at the high QC level of the 695
nominal concentration in at least 80% of the individual sources evaluated.
696
Selectivity should be evaluated in lipaemic samples and haemolysed samples (Refer to Section 697
3.2.1). For lipaemic and haemolysed samples, tests can be evaluated once using a single source 698
of matrix. Selectivity should be assessed in samples from relevant patient populations. In the 699
case of relevant patient populations there should be at least five individual patients.
700
4.2.3 Calibration Curve and Range 701
The calibration curve demonstrates the relationship between the nominal analyte concentration 702
and the response of the analytical platform to the analyte. Calibration standards, prepared by 703
spiking matrix with a known quantity of analyte, span the calibration range and comprise the 704
calibration curve. Calibration standards should be prepared in the same biological matrix as the 705
study samples. The calibration range is defined by the LLOQ, which is the lowest calibration 706
standard, and the ULOQ, which is the highest calibration standard. There should be one 707
calibration curve for each analyte studied during method validation and for each analytical run.
708
A calibration curve should be generated with at least 6 concentration levels of calibration 709
standards, including LLOQ and ULOQ standards, plus a blank sample. The blank sample 710
should not be included in the calculation of calibration curve parameters. Anchor point samples 711
at concentrations below the LLOQ and above the ULOQ of the calibration curve may also be 712
used to improve curve fitting. The relationship between response and concentration for a 713
calibration curve is most often fitted by a 4- or 5-parameter logistic model if there are data 714
points near the lower and upper asymptotes, although other models may be used with suitable 715
justification.
716
A minimum of 6 independent runs should be evaluated over several days considering the factors 717
that may contribute to between-run variability.
718
The accuracy and precision of back-calculated concentrations of each calibration standard 719
should be within ±25% of the nominal concentration at the LLOQ and ULOQ, and within ±20%
720
at all other levels. At least 75% of the calibration standards excluding anchor points, and a 721
minimum of 6 concentration levels of calibration standards, including the LLOQ and ULOQ, 722
should meet the above criteria. The anchor points do not require acceptance criteria since they 723
are beyond the quantifiable range of the curve.
724
The calibration curve should preferably be prepared using freshly spiked calibration standards.
725
If freshly spiked calibration standards are not used, the frozen calibration standards can be used 726
within their defined period of stability.
727
4.2.4 Accuracy and Precision 728
4.2.4.1 Preparation of Quality Control Samples 729
The QCs are intended to mimic study samples and should be prepared by spiking matrix with 730
a known quantity of analyte, stored under the conditions anticipated for study samples and 731
analysed to assess the validity of the analytical method.
732
The dilution series for the preparation of the QCs should be completely independent from the 733
dilution series for the preparation of calibration standard samples. They may be prepared from 734
a single stock provided that its accuracy has been verified or is known. The QCs should be 735
prepared at a minimum of 5 concentration levels within the calibration curve range: The analyte 736
should be spiked at the LLOQ, within three times of the LLOQ (low QC), around the geometric 737
mean of the calibration curve range (medium QC), and at least at 75% of the ULOQ (high QC) 738
and at the ULOQ.
739
4.2.4.2 Evaluation of Accuracy and Precision 740
Accuracy and precision should be determined by analysing the QCs within each run (within-741
run) and in different runs (between-run). Accuracy and precision should be evaluated using the 742
same runs and data.
743
Accuracy and precision should be determined by analysing at least 3 replicates per run at each 744
QC concentration level (LLOQ, low, medium, high, ULOQ) in at least 6 runs over 2 or more 745
days. Reported method validation data and the determination of accuracy and precision should 746
include all results obtained, except those cases where errors are obvious and documented.
747
Within-run accuracy and precision data should be reported for each run. If the within-run 748
accuracy or precision criteria are not met in all runs, an overall estimate of within-run accuracy 749
and precision for each QC level should be calculated. Between-run (intermediate) precision and 750
accuracy should be calculated by combining the data from all runs.
751
The overall within-run and between-run accuracy at each concentration level should be within 752
±20% of the nominal values, except for the LLOQ and ULOQ, which should be within ±25%
753
of the nominal value. Within-run and between-run precision of the QC concentrations 754
determined at each level should not exceed 20%, except at the LLOQ and ULOQ, where it 755
should not exceed 25%.
756
Furthermore, the total error (i.e., sum of absolute value of the errors in accuracy (%) and 757
precision (%)) should be evaluated. The total error should not exceed 30% (40% at LLOQ and 758
ULOQ).
759
4.2.5 Carry-over 760
Carry-over is generally not an issue for LBA analyses. However, if the assay platform is prone 761
to carry-over, the potential of carry-over should be investigated by placing blank samples after 762
the calibration standard at the ULOQ. The response of blank samples should be below the 763
LLOQ.
764
4.2.6 Dilution Linearity and Hook Effect 765
Due to the narrow assay range in many LBAs, study samples may require dilution in order to 766
achieve analyte concentrations within the range of the assay. Dilution linearity is assessed to 767
confirm: (i) that measured concentrations are not affected by dilution within the calibration 768
range and (ii) that sample concentrations above the ULOQ of a calibration curve are not 769
impacted by hook effect (i.e., a signal suppression caused by high concentrations of the analyte), 770
whereby yielding an erroneous result.
771
The same matrix as that of the study sample should be used for preparation of the QCs for 772
dilution.
773
Dilution linearity should be demonstrated by generating a dilution QC, i.e., spiking the matrix 774
with an analyte concentration above the ULOQ, analysed undiluted (for hook effect) and 775
diluting this sample (to at least 3 different dilution factors) with blank matrix to a concentration 776
within the calibration range. For each dilution factor tested, at least 3 runs should be performed 777
using the number of replicates that will be used in sample analysis. The absence or presence of 778
response reduction (hook effect) is checked in the dilution QCs and, if observed, measures 779
should be taken to eliminate response reduction during the analysis of study samples.
780
The calculated concentration for each dilution should be within ±20% of the nominal 781
concentration after correction for dilution and the precision of the final concentrations across 782
all the dilutions should not exceed 20%.
783
The dilution factor(s) applied during study sample analysis should be within the range of 784
dilution factors evaluated during validation.
785
4.2.7 Stability 786
Stability evaluations should be carried out to ensure that every step taken during sample 787
preparation, processing and analysis as well as the storage conditions used do not affect the 788
concentration of the analyte.
789
The storage and analytical conditions applied to the stability tests, such as the sample storage 790
times and temperatures, sample matrix, anticoagulant, and container materials should reflect 791
those used for the study samples. Reference to data published in the literature is not considered 792
sufficient. Validation of storage periods should be performed on stability QCs that have been 793
stored for a time that is equal to or longer than the study sample storage periods.
794
Stability of the analyte in the studied matrix is evaluated using low and high concentration 795
stability QCs. Aliquots of the low and high stability QCs are analysed at time zero and after the 796
applied storage conditions that are to be evaluated. A minimum of three stability QCs should 797
be prepared and analysed per concentration level/storage condition/timepoint.
798
The stability QCs are analysed against a calibration curve, obtained from freshly spiked 799
calibration standards in a run with its corresponding freshly prepared QCs or QCs for which 800
stability has been proven. While the use of freshly prepared calibration standards and QCs is 801
the preferred approach, it is recognised that in some cases, for macromolecules, it may be 802
necessary to freeze them overnight. In such cases, valid justification should be provided and 803
freeze-thaw stability demonstrated. The mean concentration at each level should be within 804
±20% of the nominal concentration.
805
Since sample dilution may be required for many LBA assays due to a narrow calibration range, 806
the concentrations of the study samples may be consistently higher than the ULOQ of the 807
calibration curve. If this is the case, the concentration of the stability QCs should be adjusted, 808
considering the applied sample dilution, to represent the actual sample concentration range.
809
As mentioned in Section 3.2.8, the investigation of stability should cover bench top (short-term) 810
stability at room temperature or sample preparation temperature and freeze-thaw stability. In 811
addition, long-term stability should be studied.
812
For chemical drugs, it is considered acceptable to extrapolate the stability at one temperature 813
(e.g., -20°C) to lower temperatures (e.g., -70°C).
814
For biological drugs, it is acceptable to apply a bracketing approach, e.g., in the case that the 815
stability has been demonstrated at -70°C and at -20°C, then it is not necessary to investigate the 816
stability at temperatures in between those two points at which study samples will be stored.
817
4.3 Study Sample Analysis 818
The analysis of study samples can be carried out after validation has been completed however 819
it is understood that some parameters may be completed at a later stage (e.g., long-term 820
stability). By the time the data are submitted to a regulatory authority, the bioanalytical method 821
validation should have been completed. The study samples, QCs and calibration standards 822
should be processed in accordance with the validated analytical method. Refer to Table 1 for 823
expectations regarding documentation.
824
4.3.1 Analytical Run 825
An analytical run consists of a blank sample, calibration standards at a minimum of 6 826
concentration levels, at least 3 levels of QCs (low, medium and high) applied as two sets (or at 827
least 5% of the number of study samples, whichever is higher) and the study samples to be 828
analysed. The blank sample should not be included in the calculation of calibration curve 829
parameters. The QCs should be placed in the run in such a way that the accuracy and precision 830
of the whole run is ensured taking into account that study samples should always be bracketed 831
by QCs.
832
Most often microtitre plates are used for LBAs. An analytical run may comprise of one or more 833
plate(s). Typically, each plate contains an individual set of calibration standards and QCs. If 834
each plate contains its own calibration standards and QCs then each plate should be assessed 835
on its own. However, for some platforms the sample capacity may be limited. In this case, sets 836
of calibration standards may be placed on the first and the last plate, but QCs should be placed 837
on every single plate. QCs should be placed at least at the beginning (before) and at the end 838
(after) of the study samples of each plate. The QCs on each plate and each calibration curve 839
should fulfil the acceptance criteria (Refer to Section 4.3.2). For the calculation of 840
concentrations, the calibration standards should be combined to conduct one regression analysis.
841
If the combined calibration curve does not pass the acceptance criteria the whole run fails.
842
4.3.2 Acceptance Criteria for an Analytical Run 843
Criteria for the acceptance or rejection of an analytical run should be defined in the protocol, in 844
the study plan or in an SOP. In the case that a run contains multiple batches, acceptance criteria 845
should be applied to the whole run and to the individual batches. It is possible for the run to 846
meet acceptance criteria, even if a batch within that run is rejected for failing to meet the batch 847
acceptance criteria.
848
The back-calculated concentrations of the calibration standards should be within ±20% of the 849
nominal value at each concentration level, except for the LLOQ and the ULOQ, for which it 850
should be within ±25%. At least 75% of the calibration standards, with a minimum of 6 851
concentration levels, should fulfil this criterion. This requirement does not apply to anchor 852
calibration standards. If more than 6 calibration standards are used and one of the calibration 853
standards does not meet these criteria, this calibration standard should be rejected and the 854
calibration curve without this calibration standard should be re-evaluated and a new regression 855
analysis performed.
856
If the rejected calibration standard is the LLOQ, the new lower limit for this analytical run is 857
the next lowest acceptable calibration standard of the calibration curve. If the highest calibration 858
standard is rejected, the new upper limit for this analytical run is the next acceptable highest 859
calibration standard of the calibration curve. The new lower and upper limit calibration standard 860
will retain their original acceptance criteria (i.e., ±20%). The revised calibration range should 861
cover all QCs (low, medium and high). The study samples outside of the revised assay range 862
should be reanalysed.
863
Each run should contain at least 3 levels of QCs (low, medium and high). During study sample 864
analysis, the calibration standards and QCs should mimic the analysis of the study sample with 865
regard to the number of wells used per study sample. At least 2/3 of the QCs and 50% at each 866
concentration level should be within ±20% of the nominal value at each concentration level.
867
Exceptions to these criteria should be justified and predefined in the SOP or protocol.
868
The overall mean accuracy and precision of the QCs of all accepted runs should be calculated 869
at each concentration level and reported in the analytical report. In the case that the overall 870
mean accuracy and/or precision exceeds 20%, additional investigations should be conducted to 871
determine the cause(s) of this deviation. In the case of comparative BA/BE studies it may result 872
in the rejection of the data.
873
4.3.3 Calibration Range
4.3.3 Calibration Range