Biomarker Validation Milestones

BAQ	BAQ (BMx Assay Qualification) (former BTV)	BMx Assay Technically validated assay in human matrix
	BMx Calibration Studies → BMx Candidate Selection	1) Assay of clinical samples from patients vs. age/sex-matched controls (PS, DP BMx) 2) In vivo calibration study to assess compound effects on BMx in relation to its effects on disease pathology (= IVC of the BMx) – with PJ team (TE/PD/DP)
CS	BCS (BMx Candidate Selected)	Approved by PQR & RLT as ready for clinical use
	Human qualification	Phase 1 study with exploratory BMx as a GNG
GNG to P2/3	BFP (BMx "Fit-for-Purpose)	Approved by GPT as ready for CLIA certification

to BAF

• Pilot assay established (LC/MS or LBA with appropriate reference standard)
• Buffer-based LLOQ determined with assay window

to BAQ

• Translatable human matrix available
• Detection feasibility in matrix of interest (CSF/plasma etc)
• Acceptable spike-recovery / parallelism
• Acceptable dilution linearity

to BCS

• Patient specimens available with appropriate control
• (TE/PE) Robust quantitation in individual sample with their variability
• (DR/DP, PS) Appropriate effect size in disease samples

to BCS (IVC)

• Animal model available
• Assay for pre-clinical species available
• Compound-driven modulation confirmed with PK/PD analysis

Validation

Biomarker type-specific definition of milestones

Asset:      TE -- PS LGE -- PE -- CN -- CS -- EDE IND -- EPOC →
Biomarker:        BPS  --   BFA  --  BTV  --  BCS         BCS
                                            TE, PD, DR    PS

	BPS – at Asset PS Biomarker Platform Start	BFA – at Asset PE Biomarker Feasibility Assessment	BTV – at Asset CN Biomarker Technical Validation	BCS – at Asset CS Biomarker Candidate Selection
Common	• White paper rationale • Develop BM "one-pager"	• Compatibility of reagents, assay format • Determine assay sensitivity in samples • internal vs external execution	• In vitro / in vivo evaluation of BM • Sensitivity, specificity, • Reproducibility, linearity, etc.	• In vivo Calibration (IVC) • Longitudinal response of BM in preclinical or human EM study
Molecular Biomarker				Preclinical PK-PD
PET ligand		BMax for novel PET tracer	Specific binding in rodent	All the work finished for NHP study
Neurocircuitry biomarker		Ability collect in rodent/NHP/human	Define sensitivity and selectivity in rodent/HP/human	Quantitative validation in rodent/HP/human Change is observed I patients/disease model.

Takeda Pharmaceutical Con

Asset Milestone		PS (LGE 아님!)	PE	CN	CS
Preclinical
	White paper rationale Develop BM "one-pager"	[Buffer] Compatibility of reagents, assay format Determine assay sensitivity in samples Pre-measurement experiments (calibration standard curve): spike-and recovery, Linearity of dilution (MRD: Minimum required dilution) Js: this should be done in the same matrix that we intend to develop the assay for	In vitro / in vivo evaluation of BM Sensitivity, specificity, Reproducibility, linearity, etc., (20200521 SATO SAN) method development like the established GlcSph assay from non-clinical state, and support to check further method qualification, Cf) In vitro: If CSF BM is the deliverable, we should study in supernatant (not lysate) NHP study (timing may vary): purpose: interindividual difference at BL and Tx response -Longitudinal response of BM in preclinical or human EM study	(Laura) to define Ceff as accurately as possible and choose appropriate doses for GLP Tox, using the actual development compound (the exact CN compound that would progress to CS and GLP Tox studies) & translatable readouts (&the exact translational bm we intend to evaluate in the clinic) before CS IVC Preclinical PK-PD In vivo calibration	Method transfer to CBID (이후 6m설린다고 함) (Method validation 이 아니므로, DMPK 일아닌데), Human dose prediction 위해 DMPK 에서 PK-PD correlation 분석 수행중.	(sato 상에 따르면, 이 때에 TV in clinical sample 로 보이는데) (clinical sample 에서는 human 이고, disease condition 이니까, range of biomarker value 가 다를 수 있음!)	이 단계를 clinical validation이라고 하면 안 됨 (clinical validation 은 아래definition 참고, 주로 clinical trial에서 하는 것이고, 이 단계는 여전히 analytical validation 을 clinical sample 하는 것뿐임)
activity	MBM
responsiblity			DMPK, DDU (각각 위 선 과성 했음)	DMPK
	PET ligand		BMax for novel PET tracer	Specific binding in rodent	All the work finished for NHP study
	NCBM		Ability collect in rodent/NHP/human	Define sensitivity and selectivity in rodent/HP/human	Quantitative validation in rodent/HP/human Change is observed I patients/disease model

제목으로는 1. In vivo calibration (PK-PD correlation, by DMPK) 과 2. TV in clinical sample (by CBID) 동시에 진행되나? → 아니겠네. 1 이 CS 전에 되어야 하니까, & CS 때에 transfer to CBID 한다니까 (SATO 상)

Sato Sho

(20200604) “established” includes ” ready for semi-quantification in preclinical species” in the earlier discovery stage until PE-CS, but should be used as ” ready for use in clinical trial” after CS. Most of us use establishment ambiguously. If you specify the terminology across Takeda, it would be great for us. Also I guess validation equal to ” ready for use in clinical trial” needs robustness i.e sample stability for 3~6 months and reproducibility of the measurement, so “optimization” is proba… needed for activity and mRNA assays at such an ealier stage. Be careful because these are my personal feelings.

Sho Sato: “Establishment” of GlcCer and GlcSph means establishment of methods for detection in non-clinical and clinical samples (Human brain and CSF with thy donors). Since the current method is used for discovery purpose, the method validation is needed before entry of clinical trial

20191004 DMPK activities for Acetylated Tubulin analysis

HDAC6, molecular biomarkers (10/4) DMPK, Hisao Shimizu

Purpose

• To develop CSF Acetylated Tubulin assay by using LC/MS

Progress

• Identify HDAC6 responsive novel acetylation site in beta-tubulin (K362) -completed
• Feasibility of targeted assay for acetylated beta-tubulin in mouse brain by immunocapture nano-LC/MS -on-going Once the assay is established, we will check the feasibility for the detection of the marker in CSF (rat &

Uncertain Spans

location	text/status	reason
BFP row	BFP (BMx "Fit-for-Purpose)	Closing quote/parenthesis is asymmetric in source; preserved as written.
Sato Sho block	"optimization" is proba...	Word continues past the right edge of the visible page; full token cut off.
Lower table NCBM cell	Change is observed I patients/disease model	The character before patients reads as I (likely intended in or n); preserved as visible glyph.
Lower table responsibility row	각각 위 선 과성 했음	Korean note is faintly handwritten; 과성 may be 과정.
Bottom of page	feasibility CSF sentence	Sentence continues into the next photo: “…for the detection of the marker in CSF (rat &…”