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
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
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
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)