Considerations for Clinical Pharmacokinetic Assays When Moving
from Healthy Volunteers to Disease-State Populations
Sam Willcox1, Justine Collet2, Florence Guilhot2, Graeme Evans1, Ian Skitt1 and Robert Nelson2
1Covance Laboratories Ltd, Harrogate, UK; 2Novimmune SA, Geneva, Switzerland
Introduction
Disease-State Matrix
During the clinical drug development stage, moving from Phase I firstin-human, healthy volunteer studies into Phase II/III disease-state
population studies is a critical stage for a molecule. The focus has
moved from a safety-first perspective to an efficacy, effectiveness and
safety perspective. Biological matrices such as human serum/plasma
contain a large mix of complex circulating proteins, including
components such as hormones, antibodies, electrolytes and more
(Figure 1). The composition of human serum can vary wildly between
each individual in the healthy population (sex, race, age) and even
more so in disease-state populations. This can have a large impact on
clinical pharmacokinetic (PK) assays, as it is important that clinical PK
assays are developed and validated using matrix appropriate for the
destined dosed population, be it normal healthy volunteers (NHV) in
Phase I studies or disease-state individuals in Phase II/III studies.
Moving the method into supporting the Phase II/III studies
required the introduction of the disease-state population
matrix, which can cause problems due to the additional
variable factors that occur. These can include, but are not
limited to:
▶ Increase/decrease soluble drug target
▶ Increase/decrease in endogenous target ligands
▶ High frequency of co-medications
▶ Increase/decrease or abnormal protein components
▶ Autoimmunity
▶ Higher hormone/cytokine interference
The results when moving into disease-state serum were
good, comparisons were made back-calculating diseasestate QC samples from a NHV serum calibration curve and
vice-versa. The overall inter-assay QC comparability between
the disease-state and NHV matrices was good. We also
compared the overall inter-assay QC results from the Phase I
study (Figure 6).
15625.00
Concentration (ng/mL)
Increasingly, a decision is made to cross-validate all
parameters using both NHV and disease-state serum when
moving into Phase II/III. This provides a robust set of data
for comparison and means NHV matrix may be used for the
preparation of calibrators and QC samples within the
sample analysis, reducing costs and matrix sourcing
difficulties associated with disease-state matrix.
Moving into Disease-State Matrix
Case Study Information
This case study follows the development and validation of a PK assay
for a monoclonal antibody (mAb) therapeutic in support of the Phase I
and Phase II/III clinical trials for this molecule. The initial development
activities were performed in-house by Novimmune and used the
Gyrolab platform.
As we moved into the Phase II/III development, a decision
was taken to increase the lower limit of quantification (LLoQ)
of the assay from 4.00 to 8.00 ng/mL. This decision was
taken because matrix effects are typically more pronounced
in the lower concentrations of an assay and often more so
in the disease-state. During the validation of the Phase I
study, the precision and accuracy of the LLoQ was
acceptable but a little higher than desired
(Figure 4). Due to the introduction of the disease-state
matrix and because such a level of sensitivity was not
required, the LLoQ was raised and the range of the
assay modified.
Mean Concentration (ng/mL)
Further parameters assessed in disease-state serum performed well. All
assessments passed the target acceptance criteria, with some improvements on
the Phase I assay, such as the dilution linearity (Table 1). In the selectivity
assessment for the Phase III assay 80% of individuals met target criteria
compared to 95% originally, perhaps reflecting the increased heterogeneity in the
disease-state population; however this was still within the acceptable range. The
validated parameters for the Phase III assay are listed below:
Assay Range: 8.00-6900.00 ng/mL
Intra/Inter-Assay Accuracy and Precision: Pass: All QC levels within ±20 %RE of nominal
concentrations and within ±20% precision
2
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Table 1. Dilutional Linearity Assessment of NI-0101 in Human
Disease-State Serum Across Multiple Batches
18
During the in-house development at Novimmune, poorer
precision and accuracy was noted at the upper end of the
calibration curve for the upper limit of quantification (ULoQ)
standard and the top anchor point (6900 and 20000 ng/mL,
respectively). Truncating the upper range slightly did not
improve the robustness. A comparison of the signal
amplification (PMT) settings was performed; the setting
used in the Phase I assay (5%) was compared against a
setting with lower sensitivity in fluorescence detection (1%).
Figure 5 shows an overall reduction in relative error (RE)
with the reduction in signal amplification settings.
Standard Analysis - PMT 1%
30.00
Run 01
Run 02
20.00
Run 03
Run 04
Accuracy (%RE)
10.00
0.00
4.00
8.00
16.00
32.00
102.50
282.76
820.00
2500.00
6900.00
20000.00
-10.00
-20.00
-30.00
NI-0101 concentration (ng/mL)
Standard Analysis - PMT 5%
30.00
Run 01
Run 02
20.00
Run 03
Run 04
Accuracy (%RE)
10.00
Phase I Assay Parameters
0.00
4.00
8.00
16.00
32.00
102.50
282.76
820.00
2500.00
6900.00
20000.00
-20.00
-30.00
NI-0101 concentration (ng/mL)
Figure 5. Relative error of calibration curve concentrations across multiple
runs when analysed with two different photo multiplier tube (PMT) settings.
Assay Range: 4.00-6900.00 ng/mL
Intra/Inter-Assay Accuracy and Precision: Pass: All QC levels within ±20% of
nominal concentrations and within ±20% precision
Phase II/III Assay Parameters
Room Temperature Stability: 24 hours
Moving the assay into Phase II/III required the following
parameters to be re-assessed:
Selectivity: Pass: 95.00% of individuals within ±20 %RE of nominal
concentration at LoQC and HiQC
Long-Term Freezer Stability: 12 months
ULoQ
Long-Term Freezer Stability: 3 months (time points up to 18 months under assessment)
3
-10.00
Dilutional Linearity: 1 in 2,560 dilution (in addition to 1 in 4 MRD)
HQC
1
The development strategy adopted by Novimmune was to develop and
validate an assay to support the Phase I clinical trial and therefore get
a fully functional assay in NHV matrix before assessing the disease
population. This is a common step-wise strategy adopted by the
industry; however, it requires most, if not all assay parameters to be
re-assessed within a new development/validation study before Phase
II/III studies take place. It is important to consider the later stage work
at this point to make sure the assay format used is transferable into the
relevant disease-state. An alternative strategy is to begin the assay
development including matrix from the Phase II/III disease-state
population. This often makes the initial costs higher and is a riskier
approach as there are no guarantees a compound will progress
beyond Phase I.
Freeze/Thaw Stability: 5 additional cycles
MQC
Selectivity: Pass: 80.00% of individuals within ±20 %RE of nominal concentration at LoQC
and HiQC
4
Figure 4. LLoQ (4 ng/mL) mean concentrations in relation
to %bias during the Phase I first-in-human study.
Novimmune was successful in developing an assay in NHV serum
in-house. This assay was then successfully transferred into and
validated at Covance with the following parameters achieved:
LLoQ (LQC PhI)
Figure 6. Overall inter-assay quality control mean concentrations prepared in
different matrices over multiple phases.
Inter-Assay LLoQ Duplicate
Development Strategy
8.88
9.36,
(0.91 SD) (0.63 SD)
7.88
(0.75 SD)
Dilutional Linearity: 1 in 25,600 dilution (in addition to 1 in 4 MRD)
5
1
Figure 3. Gyrolab Xp workstation.
212.24
(18.78 SD)
25.00
Quality Control Level
0
Figure 2. Gyrolab sandwich
immunoassay design.
234.71
(20.72 SD)
125.00
Freeze/Thaw Stability: 6 additional cycles
6
The standard microtitre plate sandwich format (Figure 2) is followed by
the Gyrolab platform with each step, capture-analyte-detection
(Figure 3) added consecutively but separated by wash cycles. The
platform uses fluorescence for analyte detection using a laser to excite
detection reagents tagged with Alexa Fluor® 647. Due to the Gyrolab’s
nanolitre scale format results can often be achieved within 1 hour.
226.48
(23.88 SD)
Room Temperature Stability: 24 hours
7
Gyrolab™ Platform
Healthy Serum PhII
625.00
5.00
7190.64
7032.34
(553.51 SD)
(504.03 SD)
5343.26
6660.77
(664.02 SD)
(736.57 SD)
5702.69
(381.25 SD)
Healthy Serum PhI
3125.00
1.00
Figure 1. Plasma protein composition, A) high-abundance proteins; B) moderate- and
low-abundance proteins.
5413.21
(380.98 SD)
Disease-State Serum (PhII/III)
▶ Intra/inter-assay accuracy and precision of QC
samples performed in disease-state/NHV serum
▶ Matrix effects performed in disease-state serum
▶ Room temperature and freeze-thaw stability
performed in disease-state serum
▶ Long-term freezer stability performed in
disease-state serum
▶ Dilutional linearity performed in disease-state serum
Presented at the Gyrolab User Seminar, London, June 8-9th, 2015
BackObserved
Calculated
concentration concentration
(ng/m L)
(ng/m L)
BackObserved
Calculated
concentration concentration
(ng/m L)
(ng/m L)
BackObserved
Calculated
concentration concentration
(ng/m L)
(ng/m L)
DL Sam ple
Concentration
(ng/m L)
DL Sam ple
Level
Dilution
(1 in X)
107,000.00
DL-A
5
>6 900.00
21,400.00
DL-B
25
>6 900.00
-
-
>6 900.00
-
-
>6 900.00
-
-
10,700.00
DL-C
50
>6 900.00
-
-
>6 900.00
-
-
>6 900.00
-
-
5,350.00
DL-D
100
4,716.69
471,669.00
-11.8
5,401.55
540,155.00
1.0
5,432.45
543,245.00
1.5
-
Bias (%)
-
>6 900.00
-
Bias (%)
-
>6 900.00
Bias (%)
-
-
1,337.50
DL-E
400
1,220.81
488,324.00
-8.7
1,430.66
572,264.00
7.0
1,351.43
540,572.00
1.0
334.38
DL-F
1600
323.02
516,832.00
-3.4
356.79
570,864.00
6.7
325.95
521,520.00
-2.5
83.59
DL-G
6400
85.90
549,760.00
2.8
88.38
565,632.00
5.7
78.62
503,168.00
-5.9
20.90
DL-H
25600
21.82
558,592.00
4.4
22.08
565,248.00
5.6
18.33
469,248.00
-12.3
4.18
DL-I
128000
<8.00
-
-
<8.00
-
-
<8.00
-
-
-
-
-
517,035.40
-
-
562,832.60
-
-
515,550.60
-
Mean (ng/mL)
Standard
deviation (n-1)
-
-
-
37,684.04
-
-
13,051.81
-
-
30,520.25
-
Precision (%)
-
-
-
7.3
-
-
2.3
-
-
5.9
-
Bias (%)
-
-
-
-3.4
-
-
5.2
-
-
-3.6
-
Conclusion
This case study demonstrates many of the key considerations when moving an
assay from normal healthy volunteers into the disease-state population.
It shows an assay can be fit-for-purpose for both Phase I assessment and
Phase III sample analysis with only minor adjustments; in this case, truncating
the calibration range slightly and modifying the PMT fluorescence detection
settings.
However, some assays do require major modification or even complete
re-development when moving into Phase II/III and in some cases challenges
encountered can only be solved with reagent, format and/or platform changes.
In addition, sample pre-treatment steps and specific blocking agents may need to
be included due to interfering factors within the disease-state matrix. In this study,
the speed and flexibility of the Gyrolab platform helped to achieve a broad
dynamic range, minimized matrix effects, and reduce hands-on time and cost.