Development and validation of a novel ICP-MS method to quantify different copper

species in human plasma from patients with Wilson disease

T. LIANG , H. ZHANG , L. ZHANG , S. MOSELEY , P. GUO , M. CHEN , T. HALL , M. LI , E. SWENSON , W.-J. PAN , B. MELTZER , R. PELTO and M. MA
1 2 3 3 2 3 3 3 3 3 3 3 1

1
Previously employed* by Alexion, AstraZeneca Rare Disease, Boston, MA, USA; 2Frontage Laboratories, Inc., Exton, PA, USA; 3Alexion, AstraZeneca Rare Disease, Boston, MA, USA
*Research carried out while employed by Alexion, AstraZeneca Rare Disease, Boston, MA, USA
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INTRODUCTION RESULTS
• Ceruloplasmin (CP) is a major copper-carrying protein in the blood; CP-bound • Currently, assays recommended in guidelines from the American Association of Liver
Method validation Clinical sample testing
copper (CP–Cu) is considered to be nontoxic because it is nonexchangeable at Disease and the European Association for the Study of the Liver indirectly estimate the
• Full validations were successfully performed for each copper fraction, as well as for • The CP–Cu:CP molar ratio observed differed from the predicted ratio of six; the mean
physiological pH, whereas non-CP-bound copper (NCC) is exchangeable and therefore level of NCC by subtracting the concentration of CP–Cu from the total serum copper
CP, and demonstrated a linear range of 5 to 1000 ng/mL (0.08 to 15.75 µM) for CP–Cu, (standard deviation [SD]) was 4.68 (0.631) in healthy volunteers and 3.42 (3.089) in
potentially reactive.1,2 concentration (calculated NCC [cNCC]).5,7 This method assumes that six copper atoms
dNCC and LBC, and of 5000 to 800 000 ng/mL (0.04 to 5.97 µM) for CP.
• In healthy individuals, approximately 90% of copper in plasma is bound to CP.3 are bound to each CP molecule; in reality, the ratio may vary.8 patients with WD (Table 2).
• The precision and accuracy of intra- and inter-run comparisons met the predefined

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• In patients with Wilson disease (WD) – a rare, autosomal recessive condition – defective • In addition, the existing guideline-recommended method may not accurately capture • Mean concentrations of CP–Cu and of plasma total copper were lower in patients with
ATP7B leads to inadequate loading of copper into CP, resulting in accumulation NCC levels in patients being treated with ALXN1840. acceptance criteria for quality control (Table 1).
WD than in healthy volunteers (Table 3).
of copper in the tissues and an increase in exchangeable copper, or NCC, in – ALXN1840 (bis-choline tetrathiomolybdate [TTM], formerly named WTX101) is an • Injection carryover for the analyte and the internal standard was within acceptable
thresholds for validation (data not shown). • In patients with WD who were enrolled in the phase 3 study of ALXN1840, 22% had
the blood.3,4 investigational copper-binding agent that mobilizes blood and tissue copper and has
• In the selectivity evaluation, all individual plasma lots were within the accepted criteria negative cNCC values at baseline. For patients with positive values (n = 162), the
– Successful treatment of WD relies on agents that can remove excess copper from demonstrated a significant NCC-lowering effect in a phase 2 study.9
the body.5 – The mode of action of ALXN1840 is unique; it forms an inert TTM–albumin–copper for validation (100% ± 20% of the nominal concentration for each analyte). mean (SD) baseline cNCC concentration was 2.06 (1.65) µmol/L.
• All stability measures were also acceptable for validation (Table 1).

THE INTERNATIONAL
– Standard of care (SoC) involves removal of excess copper using metal chelators and tripartite complex (TPC) that does not contribute to the exchangeable copper pool.9,10
Table 2. Molar ratio of CP–Cu:CP in patients with WD and in healthy volunteers
limiting copper absorption using zinc; however, in many patients, symptoms persist To calculate NCC in ALXN1840-treated patients, the TPC would need to be measured

TM
Table 1. Intra- and inter-run validation parameters
or worsen.6 and subtracted from the NCC fraction.9 A direct assay is therefore needed.

LIVER CONGRESS
CP–Cu:CP ratio Patients with WD Healthy volunteers
dNCC and LBC CP
(n = 207) (n = 17)
CP–Cu
Mean (SD) 3.42 (3.089) 4.68 (0.631)
Volume of human lithium 20 20 20
Median (Q1, Q3) 2.93 (2.36, 3.49) 4.68 (4.31, 5.05)
AIM plasma sample, μL
Platform ICP-MS ICP-MS LC-MS/MS CP, ceruloplasmin; CP–Cu, ceruloplasmin-bound copper; Q1, quartile 1; Q3, quartile 3; SD, standard
deviation; WD, Wilson disease.
• To develop and validate a novel assay that isolates multiple copper species (CP–Cu, directly measured NCC [dNCC] and labile bound copper [LBC]) from plasma. Quantitation range, ng/mL (µM) 5 to 1000 5 to 1000 5000 to 800 000
(0.08 to 15.75) (0.08 to 15.75) (0.04 to 5.97)
Table 3. Concentrations of CP–Cu, dNCC and LBC in patients with WD and in healthy
QC intra-run precision, %CVa 1.1 to 16.8 1.1 to 14.4 2.9 to 13.3
volunteers
QC intra-run accuracy, %bias a
–15.5 to 8.7 –19.7 to 18.6 –16.0 to 16.0

METHOD QC inter-run precision, %CVa

QC inter-run accuracy, %biasa
1.9 to 10.3
–10.3 to 5.6
1.9 to 9.6
–4.4 to 12.0
7.2 to 9.8
–14.0 to 7.4
Patients with WD
(n = 207)
Healthy volunteers
(n = 17)
Figure 1. Summary of novel assay processa Sample bench-top stability at 19 17.5 19 CP–Cu concentration, µM, 3.03 (3.33) 11.30 (1.72)
room temperature, hours mean (SD)
(a) (b) Immunocapture Sample short-term stability at 19 17.5 19 dNCC concentration, µM, 1.03 (1.00) 0.51 (0.10)
Prepare anti-CP Incubate plasma Use magnetic separation 4°C ± 4°C, hours
TTM– antibody-coated sample with and remove supernatant mean (SD)
Peptide–Cu Albumin–Cu CP–Cu Freeze/thaw stability, number 4 4 4
albumin–Cu beads coated beads LBC concentration, µM, 1.04 (0.87) 0.50 (0.10)
of cycles

DOI: 10.3252/pso.eu.ILC2022.2022
Total plasma copper mean (SD)
a
Results include combined %CV or %bias from all QC levels, including LLOQ, low, matrix low, mid, matrix
Immunocapture of CP dNCC CP CP–Cu mid, high and matrix high QCs. Acceptance criteria for QC samples were: %bias within 15.0% (within 20.0% Plasma total copper 4.86 (4.13) 13.75 (1.72)
for LLOQ) and %CV ≤ 15.0% (≤ 20.0% for LLOQ); the predefined acceptance criteria in human plasma were:
%bias within 20.0% (within 25.0% for LLOQ) and %CV ≤ 20.0% (≤ 25.0% for LLOQ).
concentration, µM,
CP–Cu %CV, percentage coefficient of variation; CP, ceruloplasmin; CP–Cu, ceruloplasmin-bound copper; mean (SD)
dNCC, directly measured non-ceruloplasmin-bound copper; ICP-MS, inductively coupled plasma mass
spectrometry; LBC, labile bound copper; LC-MS, liquid chromatography–mass spectrometry; LLOQ, lower limit CP–Cu, ceruloplasmin-bound copper; dNCC, directly measured non-ceruloplasmin-bound copper;
TTM–
Peptide–Cu Albumin–Cu of quantitation; MS, mass spectrometry; QC, quality control. LBC, labile bound copper; SD, standard deviation; WD, Wilson disease.
albumin–Cu
(c) Chelation and filtration
dNCC
Chelation and filtration
Add chelation Transfer incubated 30 kDa
TTM–
albumin–Cu
solution samples filter
LBC
CONCLUSIONS
Collect • Validation experiments confirmed that this novel assay directly measuring CP–Cu, NCC, LBC and CP conforms to the accepted criteria for precision, accuracy, selectivity and stability.
Peptide–Cu Albumin–Cu filtrate
dNCC Incubate • Data also support existing evidence suggesting that the ratio of CP–Cu:CP can be lower than the theoretical value of six in healthy people and in those with WD, and that calculation of
LBC NCC using an assumption of six atoms of copper per CP molecule is not accurate.8
• This assay could potentially be used for efficacy assessment of ALXN1840 in clinical trials and may have broader utility in diagnosis and treatment monitoring in patients with WD.
Method scheme. (a) Overview of assay procedure (TTM, the active moiety of ALXN1840, which can bind to copper and albumin). (b) Immunocapture steps to obtain dNCC, CP and CP–Cu species. (c) Chelation and filtration
steps to isolate LBC.

General hepatology
a
Capture of the TTM–albumin–Cu tripartite complex is important for quantifying LBC in patients treated with ALXN1840, but it is not necessary for measuring dNCC nor relevant for LBC data from samples collected at baseline.
All samples described herein were collected at baseline.
CP, ceruloplasmin; Cu, copper; dNCC, directly measured non-ceruloplasmin-bound copper; LBC, labile bound copper; TTM, tetrathiomolybdate.
REFERENCES ACKNOWLEDGMENTS

Ryan Pelto
• The assay directly quantifies multiple copper species from human plasma using a combination of steps: immunocapture of CP using magnetic beads coated with a monoclonal 1. Twomey PJ et al. J Trace Elem Med Biol 7. European Association for Study of the Liver. Writing assistance for this poster was provided by Oxford PharmaGenesis, Oxford, UK, with funding from
antibody; chelation; filtration and inductively coupled plasma mass spectrometry (ICP-MS). These combined sequential processes enable bioanalysis of CP and various copper 2008;22:50–3. J Hepatol 2012;56:671–85. Alexion, AstraZeneca Rare Disease. Funding for the study was provided by Alexion, AstraZeneca Rare Disease.
2. Kirsipuu T et al. Sci Rep 2020;10:5686. 8. Twomey PJ et al. J Clin Pathol 2007;60:441–2.
species, including CP–Cu, dNCC and LBC, from human plasma (Figure 1). 3. Quarles CD Jr et al. Metallomics 2020;12: 9. Weiss KH et al. Lancet Gastroenterol Hepatol CONFLICTS OF INTEREST
• Guidance from the US Food and Drug Administration for methodological validation was followed, incorporating measures for precision, accuracy, selectivity and stability.11 The following 1348–55. 2017;2:869–76.
At the time the research was carried out, the authors were all either employed by Alexion
4. Patil M et al. J Clin Exp Hepatol 2013;3:321–36. 10. Brewer GJ et al. Transl Res 2009;154:70–7.
validation parameters were examined: linear range, sensitivity, intra- and inter-run precision and accuracy, selectivity, carryover and sample stabilities (freeze/thaw and short- and 5. Roberts EA et al. Hepatology 2008;47: 11. US Food and Drug Administration. Bioanalytical
Pharmaceuticals, Inc., who validated the method, or involved in Alexion-sponsored projects at
long‑term stabilities). 2089–111. method validation guidance for industry. 2018. Frontage Laboratories, Inc., during which normal reference determination was performed.
6. Appenzeller-Herzog C et al. Liver Int Available from: https://www.fda.gov/downloads/
• Plasma samples from 207 patients enrolled in the phase 3 study of ALXN1840 in WD (NCT03403205) were assessed at baseline (before treatment) using the novel assay. 2019;39:2136–52. drugs/guidances/ucm070107.pdf (Accessed CONTACT INFORMATION

FRI--268
For comparison, samples from 17 healthy volunteers enrolled in a phase 1 study (NCT04594252) were also evaluated. May 12, 2022). [email protected]

Presented at the International Liver Congress™ (ILC) 2022, 22–26 June 2022, London, UK
ILC2022