CSL730 TDM/Hemapen

Indications

CSL730

rec. trivalent hIgG1 Fc Trimer

Treatment of immune complex-mediated autoimmune diseases

Immune Thrombocytopenic Purpura (ITP)

Administered by subcutaneous injection/infusion

Premedication regimen: chlorphenamine & methylprednisolone (for cohort 1-3, unless recommended otherwise)

10 Cohorts - 6 subjects each (2 placebo & 4 subjects)

Planned doses are putative and may be adjusted based on emerging data

Testing whole blood (PK & PD)

PK: measure CSL730 concentrations

Pharmacodynamics

Flow

Whole blood: Fc gamma receptors in circ. leukocyte subsets

Soluble biomarkers

PGx

Whole blood: genetic profile

Whole blood: inhibition of FcyR activation & exploratory biomarkers

Safety markers: IgG, cytokines, inflammatory markers, complement...

Developed in collaboration with Momenta Pharmaceuticals.

Chronic inflammatory demyelinating polyneuropathy (CIDP)

Pemfigus filgarus

myasthenia gravis (MS)

DBS for TDM

Considerations

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Translation from EMA & FDA guidelines

Cross-Val.

Sample Collection

Is there any interference from the collection material itself (blank?)

Is drying a concern? Improper dryign may affect analyte stability/contamination

Incorporation of an internal standard

Matrix used for Cal. curves and internal QCs

Whole blood? Does HemaPen have an anticoagulant? Impractical to prepare spiked samples in anti-coagulated blood

If not, can we draw pre-dose aliquots from the patient for noanticoagulent/anticoagulent testing?

prep of spiked Cal. and QCs may require HT or erythrocyte volume fraction adjustment

DBS-specific Val. Parameters

MD- Stress Testing

Assess time sensitivity

IL-1Ra

IL-6

p/tSYK

Analyze 50-60C for atleast 2 days to mimic aged samples?

Other factors: humidity and exposure to light

Volume & hematocrit & homogeneity

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Stability concerns for enzymatic and oxidation sensitive analytes

For therapeutic drug monitoring: cal. range conc. range should be representative of expected patent sample conc

Suggested 412 DBS required for Validation (258 DBS if volume, HT, volcano, carryover and stability @ 60C are not assessed). Further decreases if carryover and nr. of Stability&Recovery replicates are reduced

Selectivity (n= 12) 6BLK+6LLOQ

Calibration curve (30) 6 Cal levels x 5 days

Accuracy & precision (n=24) 4 QC levels (duplicate)x 3days

Dilution Integrity (n=6) 1 QC level (duplicate) :x 3 days

Carryover (n=10) Blank & Zero sample x 5 days

Recovery/Mat effect/process efficiency (n=170)

2 QC levels x 6 donors (5 replicates)

1 donor x 3 HT levels x 2 QC levels

2 QC levels for Standard solutions (5 replicates)

Stability (n=40)

2 QCs @ 1 HT level (5 replicates)

4 Timepoints @ RT & 60C

Volume/HT/Volcano Effect (n=120)

2 QC levels @ 3 HT levels (5 replicates)

4 volumes (3x vol. levels for central punch and 1x high peripheral punch vol)

Extraction solvent spiked with QC level of standard

test for sampling contam (more an issue for PCR-based techniques)

Matuszewski approach?

Clinical Validation: Comparing DBS to Venous blood/plasma/serum

Recommended min. 40 samples from 25 subjects (for blood and DBS)

Differences in peripheral vs venous blood (distribution phase)

HT determination

Comparing DBS to venous blood/anticoagulated blood//serum/plasma

drug may bind to plasma components, accumulate in RBCs which may impact results between matrices

Analytical methods can be used to include HT correction

not necessary for HT-dependent method if HT range in that specific population is narrow & within method acceptance limits

HT effect less pronounced with fixed spot volume

Stats Interpretation

Regression analysis

weighted Deming regression

Passing-Bablok

Assess agreement/estimate bias

Bland-Altman

Decide beforehand what a clinically relevant or acceptable bias is beforehand (limits of agreement)

HemaPen

4x 3.5 mm paper discs (substrates)

55% hemacrit may impair collection

K2-EDTA-coated capillaries

~2.74 uL vol per disc

ISO 9001 certified

Requires opening tool to remove discs

Needs to be replaced often (every 150-200 hemaPENs)

Discs can be customized

typically 226 Perkin Elmer paper

Extraction

100 ul chilled methanol in 1.5 ml eppie used for metabolics

100 ul butanol:methanol (1:1) in 1.5 ml eppie for lipidomics

Literature

DBS validation for CVD biomarkers

Val. of biomarkers from DBS vsSerum. Buxton et al. 2016 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4812568/

HT determination

Potassium measurement

Spectroscopy

Sulfolyser reagetnt

noncontact diffuse reflectance

near-infrared

DBS may less of a barrier to participation

Established serum to DBS equivalence rather than comparing to whole blood

Would require single use micro-lancet & alcohol swabs

DBS hs greater variability compared to venous blood and therefore less reliable on an individual level

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DBS for TDM of IBD (Mathot et al., 2019)

Elution buffer: PBS-T/0.05%NaN3 vigorously shaking overnight (>17h) on orbital shaker. Eluate kept at 4C

HT conversion of DBS

DBS validated for measurement of adalimumab

Benefits

TDM helps maintain drug at trough concnetrations and can prevent deleterious anti-drug antibody formation

Faster turnaround time

Competitors

Mitra microsampler

Fixed HT (e.g. 0.42)

Normalised to HT timepoint 1 or most recent timepoint

Normalised to hemaglobin conc. (Abs@415 nm)

Elution efficiency

Weak but significant influence of HT on DBS

DBS has PK potential

DBS Competitor technology

Mitra Cartridges (Neoteryx)

HemaSpot

tamper resistant, moisture tight cartridge containing filter paper and dessicant

Sizes (10-30 ul)

No Volumetric Blood Hematocrit Bias

No anticoagulant?

rare autoimmune neuromuscular disorder - muscle weakness, reduced physical performance & increased muscular fatigue

Prevalence: <5 per 100 000 in EU and <1 per 500 000 in China

$2.1 billion cost of hospitalization in US between 2010-2012

Mean cost for CIDP-related hospitalization was $70 000

Health plan-paid costs ~$46-66k (2/5 medical costs and 3/5 pharmacy costs, of which ~90% for IVIg therapy) - higher than MG

Market

Hypogammaglobulinemia

Inflammatory myopathies

market estimated at $9 million in 2017 and projected $16mil by 2025.

IVIg market - >50 primary immunodeficiency diseases including 176 rare hereditary disorders

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HemaSpot

correlation between DBS and venous blood appears to be analyte specific

Most commercial assays have not been validated for use with DBS and therefore additional regulatory approval may be required

Simplified specimen transport and logistics

Annual reports

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CSL IVIG = privigen

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Quality of life

Approx. 30% of patients will progress to wheelchair dependence if not treated

FDA on DBS

Section E:Validation should address, at a minimum, the effects of the following issues: storage and handling temperatures, homogeneity of sample spotting, hematocrit, stability, carryover, and reproducibility, including ISR

Validation support literature

Standards for Reporting of Diagnostic Accuracy - DBS

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Blood collection and Handling - DBS- module

Adv/DisAvd_Description of effect (book)

Ab Stability

IgA requires further study

Extraction: IgM declines rapidly on storage, regardless of temp (not detectable at -20 after on month)

IgG relatively stable on filter paper over 4-month period at lower temp

Serology: Sera v DBS

excellent corelation

Standardized method for Ab detection in DBS

6 mm punch contains roughlt 6 ul serum

Antibodies

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IgM exists as a membrane bound monomer or secreted pentamer and is the first antibody produced in response to antigen

Correlation between CH50 and symptoms

20 in 100000 in US diagnosed each year (MG foundation of America) and 14-40 per 100000 (National Organization for Rare disorders)