analytical performance evaluation of a dedicated

Analytical performance evaluation of a

dedicated calibrator and controls for

emicizumab▼quantification

Narayanan Ramamurthy,1 Cheryl Kucharski,1 Mags McInerney,1

David Chen,2 Michael Morris1

1r2 Diagnostics, South Bend, IN, USA; 2Genentech Inc., South San Francisco, CA, USA

▼ This medicinal product is subject to additional monitoring. This will allow quick identification of new safety information. Healthcare professionals are asked to report any suspected adverse reactions.

These should be reported to the Regulatory authorities in your country according to your national requirements.

Disclosures for Narayanan Ramamurthy

2

Disclosures

Employment r2 Diagnostics

Emicizumab

– Bispecific antibody that mimics the

function of missing activated FVIII,

restoring haemostasis in PwHA1

– Much higher apparent FVIII activity than

expected with one-stage FVIII assays

MOSA for emi

– Calibration curve:

• Emi calibrator, FVIII DP, aPTT reagent

and CaCl2

– 1:8 predilution on analyser prior to assay

3

Background

Emicizumab

FIXaFX

aPTT, activated partial thromboplastin time; DP, depleted plasma; emi, emicizumab; F, factor; MOSA, modified one-stage assay;

PwHA, persons with haemophilia A1. Kitazawa T, et al. Thromb Haemost 2017;117:1348–1357.

Optimised on BCS XP (Siemens) using the

FVIII one-stage assay method

– Samples pre-diluted 1:8 on analyser

– Emi calibrator instead of standard

human plasma

– Emi controls instead of FVIII normal and

abnormal plasma controls

Reagents:

– Emi calibrator: 100 µg/mL (r2 Diagnostics)

– Emi controls: L1=25 µg/mL; L2=75 µg/mL

(r2 Diagnostics)

– Actin FSL, CaCl2 and FVIII deficient plasma

(Siemens)

Calibration range:

– 10–100 µg/mL emi

1.60

1.65

1.70

1.75

1.80

1.85

1.90

0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20

Lo

g c

lot

tim

e (

se

c)

Log [emi] (µg/mL)

Emicizumab MOSA calibration curve

4

MOSA for emicizumab

Performed per CLSI EP17-A2 guidelines:

– Samples: FVIII DP with and without spiked emi

– Reagents: 2 lots of all assay reagents

LoB:

– 5 blank samples x 4 runs/day x 3 days

LoD:

– 7 low level emi samples (2.5–17.5 µg/mL) x 4

runs/day x 3 days

LoQ:

– 7 low level emi samples x 4 runs/day x 3 days

5

Analytical sensitivity – LoB/ LoD and LoQ

Parameter Result (µg/mL)

LoB 1.8

LoD 3.2

LoQ 10.0

LoB, limit of blank; LoD, limit of detection; LoQ, limit of quantitation

CLSI EP17-A2, Clinical and Laboratory Standards Institute document EP17-A2—Evaluation of Detection

Capability for Clinical Laboratory Measurement Procedures; Approved Guideline—Second Edition.

Performed per CLSI EP06-A guidelines:

– Samples: FVIII DP spiked with emi


Method:

– Sample series (0–250 µg/mL emi) tested

– 4 runs/ sample

– Samples >100 µg/mL run with reflex testing

Results

– Lot 1 slope=0.89, R2=0.99

– Lot 2 slope=0.95, R2=1.00

– Lot 3 slope=0.93, R2=0.99

6

CLSI EP06-A, Clinical and Laboratory Standards Institute document EP06-A—Evaluation

of the Linearity of Quantitative Measurement Procedures: A Statistical Approach

Assay linearity

Performed per EP05-A3 guidelines:

– Samples:

• FVIII DP spiked with emi

(10–200 µg/mL)

• Calibrator and controls (3 lots each)

– Analysis:

• Nested ANOVA

Repeatability:

– 20 days x 2 runs/day x 4 replicates/run

– 3 lots of reagents tested (1 lab)

Results:

– Excellent precision seen across

measurement range

– Total %CV values 6.3–10.2%

7ANOVA, analysis of variance; CV coefficient variation

CLSI EP05-A3, Clinical and Laboratory Standards Institute document EP05-A3—Evaluation of

Precision of Quantitative Measurement Procedures; Approved Guideline—Third Edition

Precision – repeatability within one site

Within

run

(%CV)

Between

run

(%CV)

Between

day

(%CV)

Between

calibration

(%CV)

Between

lot (%CV)

Total

(%CV)

Spiked

samples3.4–4.0 2.0–7.0 3.0–5.2 0.0–4.0 0.0–4.9 6.3–10.2

Calibrator 3.6 1.9 3.4 4.1 0.0 6.7

Control

L13.3 3.4 2.4 1.2 2.4 6.0

Control

L23.1 3.3 1.6 4.9 0.0 6.9

8

Precision – reproducibility across three sites

Within

run

(%CV)

Between

run

(%CV)

Between

day

(%CV)

Between

lab

(%CV)

Total

(%CV)

Spiked

samples5.1–9.0 0.8–1.4 0.9–2.4 0.9–3.6 5.5–10.2

Calibrator 5.2–6.7 0.8–2.5 0.6–2.8 4.6–6.1 7.5–9.9

Control

L16.1–6.5 0.9–1.1 1.4–1.8 0.9–1.5 6.5–6.9

Control

L24.8–5.1 0.5–1.4 1.0–2.3 0.9–2.1 5.3–6.1


– Samples:

• FVIII DP spiked with emi

(10–200 µg/mL)

• Calibrator and controls (3 lots each)

– Analysis:

• Nested ANOVA

Reproducibility:

– 5 days x 2 runs/day x 3

replicates/run

– 1 reagent lot tested across 3 labs

Results:

– Excellent precision seen across

measurement range

– Total %CV values 5.3–10.2%

CLSI EP05-A3, Clinical and Laboratory Standards Institute document EP05-A3—Evaluation of

Precision of Quantitative Measurement Procedures; Approved Guideline—Third Edition



– Analysis: paired difference method


Method:

– 10, 100 and 200 µg/mL emi spiked with 4

levels of each interferent + one 0

interferent level

– Each sample tested in quadruplicate

Results

– aPCC, standard rFVIII and porcine rFVIII

falsely elevate the emi measurement

– No interference from rFVIIa and

heparin (up to 1 U/mL)

9aPCC, activated prothrombin complex concentrate; rFVIIa, activated

recombinant factor VII; rFVIII, recombinant factor VIII

CLSI EP07-A2, Clinical and Laboratory Standards Institute document EP07-A2—

Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition

Analytical specificity – exogenous interferences

Interferent Target range Effect

aPCC 0–6 U/mL Emi falsely elevated

rFVIIa 0–5 µg/mL No interference

Standard rFVIII 0–2 U/mL Emi falsely elevated

porcine rFVIII 0–2 U/mL Emi falsely elevated

Unfractionated

heparin0–10 U/mL No interference up to 1 U/mL

Maximum allowable difference for each combination ≤10%



– Analysis: paired difference method


Method:

– 10, 100 and 200 µg/mL emi spiked with 4

levels of each interferent + one 0

interferent level

– Each sample tested in quadruplicate

Results

– No interference from endogenous interferents

10CLSI EP07-A2, Clinical and Laboratory Standards Institute document EP07-A2—Interference Testing

in Clinical Chemistry; Approved Guideline—Second Edition

Analytical specificity – endogenous interferences

Interferent Target range Effect

Hemolysis

(hemoglobin)0–500 mg/dL No interference

Lipemia

(triglycerides)0–2000 mg/dL No interference

Icterus (bilirubin) 0–20 mg/dL No interference

Lupus anticoagulant

(high titer)

0–73.2 DSec StaClot

LANo interference

FVIII inhibitor

(high titer)0–108 BU/mL No interference

Maximum allowable difference for each combination ≤10%


– Patient samples: PwHA on emi treatment

Method:

– MOSA tested at 3 sites on fresh and frozen

samples (ongoing)

– Samples tested on emi specific ELISA (used

in phase 3 studies of emi)

– 146 samples tested across all sites

Results

– Slope=0.97; R2=0.9

– Excellent agreement between ELISA and

MOSA across the range 0–150 µg/mL

11

CLSI EP09-A3, Clinical and Laboratory Standards Institute document CLSI EP09-A3—Measurement Procedure

Comparison and Bias Estimation Using Patient Samples; Approved Guideline—Third Edition

Method comparison – MOSA vs ELISA

y = x

y = 0.9727 + 1.9628

R² = 0.9007

0

20

40

60

80

100

120

140

160

0 20 40 60 80 100 120 140 160

MO

SA E

mic

izu

mab

Co

nce

ntr

atio

n (μ

g/m

L)

ELISA Emicizumab Concentration (μg/mL)

Linear Regression Plot: ELISA vs MOSA All sites

Stability:

– Sample stability: emi treated PwHA plasma

– Shelf-life stability: emi calibrator and controls

– Reconstituted stability: emi calibrator

and controls

Method:

– Samples tested at T0 and at various

timepoints at each storage condition

– Timepoint results compared to T0

– Allowable drift: ±2 SD

Results:

– Sample, calibrator and controls demonstrated

acceptable stability for all conditions tested

12h, hour; m, month; RT, room temperature; SD standard deviation; w, week

Stability

Stability

type

Stability

condition

Stability

duration

Sample

RT

-20°C

-80°C

4h

up to 3w

up to 4m

Shelf-life* 2–8°C 12m

Reconstituted

20–25°C capped

8–15°C uncapped

2–8°C capped

8h

8h

24h

*Testing ongoing

A MOSA was developed along with a dedicated calibrator and controls for the measurement of

emi in plasma samples

MOSA shows good sensitivity and linearity across a measurement range of 10–250 µg/mL emi

MOSA shows good repeatability and reproducibility across a measurement range of

10–200 µg/mL (%CV 5.3–10.2)

No measurable impact on emi recovery seen in the presence of endogenous interferences

rFVIIa and unfractionated heparin (up to 1U/mL) show no effect on emi recovery

Standard rFVIII, aPCC and porcine rFVIII at prophylactic levels result in falsely elevated emi

level

MOSA shows excellent agreement with an emi-specific ELISA assay

Sample stability, calibrator and control shelf-life and reconstituted stability are acceptable

13

Summary

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