carbon-14 l abelled adcs dr william h. watters isotope chemistry manager

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Carbon-14 Labelled ADCs Dr William H. Watters

Isotope Chemistry Manager

william.watters@almacgroup.comwww.almacgroup.com

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Almac overview

BiomarkerDiscovery &Development

API Services& Chemical

Development

PharmaceuticalDevelopment

ClinicalTechnologies

ClinicalTrial Supply

AnalyticalServices

CommercialServices

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1 Small molecule development

2 Biocatalysis + Isotopic Labelling

3 Peptide and protein technology

4 Physical sciences

5 Analytical services

API services and chemical development

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14C radio labelling: API and IMP

• Non-GMP and cGMP synthesis

• API and IMP (drug product)• Small molecules, peptides and

conjugates• Dedicated API and IMP

facilities• Packaging, QP release and

dispatch to clinical trial site

Discovery of 14CMartin Kamen & Sam Ruben (27-FEB-1940)

T1/2 ~ 5730 Years

[14C]-ADME Studies• Absorption

• What fraction goes into systemic circulation

• Distribution• Does the drug reach the site of action

• Metabolism• What is the drug turned into and what it comes out as

• Excretion• How the drug is removed from the body and how fast

Choice of radiolabelRadioisotope Half Life

14C 5730 years3H 12.3 years35S 87.6 days125I 60.1 days131I 8 days32P 14.3 days33P 25.3 days

•Almost all pharmaceutical studies with small molecules are done with 14C. •14C present in the skeleton of all drug molecules.• 14C is Detectable at very low concentrations (scintillation counting)• Long half life means no need for correction for radioactive decay.•3H is also used but is more subject to exchange.

14C radiolabelling common termsCommon units used in Radiolabelling

MilliCurie (mCi), and microCurie (Ci) for quantityAlternative Units

Megabecuerels (Mbq) (1mCi = 37Mbq)Specific Activity

Commonly expressed in mCi/mmol or Ci/mgLabelling one carbon atom with 14C results in a maximum specific activity of 62.4mCi/mmol

•

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Specification:• [14C]-mAb-Protein Conjugate required carbon-14 label on the linker

• Specific Activity of ≥ 1.1 Ci/mg and 4 g of material

CASE STUDY 1:[14C]-mAb-Protein Conjugate

mAb

DRUG(Protein) 14C14C DRUG

(Protein)

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Strategy: [14C]-Linker Chemistry

Drug

mAb

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• [14C]-Linker (1 eq) reacted with Protein Drug (via maleimide linkage)

• IPC analysis by HPLC to determine completion of activation

• Reaction temperature critical to minimise degradation

• Unbound [14C]-Linker removed using DF (10 kDa membrane)

Protein Drug(10-30% disulfide)

TCEP Reduction

Protein Drug(Fully reduced)

2. UltrafiltrationProtein-linkerconjugate

14C14C1.

Step 1: Drug - [14C]Linker Activation

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• [14C]-Linker-Drug (4.8 eq) conjugated with mAb (via amide linkage)

• IPC analysis by SEC HPLC to determine completion of conjugation

• Product filtered through 0.22 µm filter to reduce bioburden

Protein-linkerconjugate

2. UF/DF3. HIC purification

[14C]-mAb-Protein Conjugate

14C

1.

14C 14C

Mwt = 180 kDa

Step 2: Antibody Conjugation

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• [14C]-mAb-Protein Conjugate purified using HIC chromatography

• Fractions collected and analysed using SEC HPLC

• Salt exchanged using DF and sample concentrated (30 kDa membrane)

• Product filtered (0.22 µm filter) and formulated in pharmacological buffer

[14C]-mAb-Protein Conjugate

14C 14C

Mwt = 180 kDa

Step 3: Purification / Formulation

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• 4.36 g [14C]- mAb-Protein Conjugate obtained

• 21% Radiochemical yield from [14C]-Linker

• Specific activity 1.20 Ci/mg (Gravimetric)

• All customer target specifications were met

• Bacterial Endotoxin levels <0.3 EU/mL

• BioBurden < 1 CFU/0.5mL

Summary: Case Study 1

Specification:

• 240 mg of [14C]-biomolecule• Specific Activity > 320 mCi/mmol

N

O

O

S

14CGly

AA-SEQUENCE LINKER PEG

mAb

CASE STUDY 2: [14C]-Biomolecule

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Stage 1: [14C]-Peptide

14CGly

AA-SEQUENCE LINKER

AA-SEQUENCE LINKER

Boc

H2N Resin

14CGly

Boc COUPLING

14CGly

AA-SEQUENCE LINKERBoc

CLEAVAGE

Resin

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N

O

O

14CGly

AA-SEQUENCE LINKER PEG

14CGly

AA-SEQUENCE LINKERBoc

N

O

O

PEGN

O

O

O

Boc

N

O

O

14CGly

AA-SEQUENCE LINKER PEG

Boc - Deprotection

Stage 2: PEGylation

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N

O

O

S

14CGly

AA-SEQUENCE LINKER PEG

mAb

N

O

O

14CGly

AA-SEQUENCE LINKER PEG

Dialysis50 kDa membrane

Stage 3: Bio-conjugation

Summary: Case Study 2• 250 mg of [14C]-biomolecule prepared• Total Protein 4.4 mg/ml• Molecular weight identity (SDS Page): equivalent to cold

standard• Stability issues with intermediate PEG peptide successfully

resolved

N

O

O

S

14CGly

AA-SEQUENCE LINKER PEG

mAb

S.L. Kitson, T.S. Moody, D.J. Quinn, A. Hay, ‘Carbon-14 Bioconjugation: Peptides andAntibody-Drug Conjugates’, Pharmaceutical Sciences, Manufacturing & Marketplace Report, May 8 (2013).

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Manufacture of Monomethyl Auristatin building blocks

Challenges:• Complex chiral chemistry• Control of chiral centres• Diastereoselective reductions• Cryogenic chemistry• Avoidance of epimerisation

Manufacture:• kg scale• Larger scale if required

(1000L reactors)

Purification:• Crystallisation

NH

HO

ON

OMeON

OMe

OHN

OHN

MMAE

NH O

OHO

BnO.HNcy2

HNOtBu

OOMeMe

Z-Ile-OH.DCHA

.HCl

NOMe

OH

OBoc

NOHBoc

Boc-Prolinol

NH

HO

ON

OMeON

OMe

OHN

OHN

MMAE

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Challenges: Solution phase peptide

chemistry Avoidance of epimerisation Physical form of products Purification

Manufacture: 100s gram scale to date larger scale if required

(50L reactors)

Purification: Biotage chromatography

(kg scale) Preparative HPLC

(15cm column)

NHO

NOMeO

NOMe

O

R

HN

O

R

HNR

RR

RMMAE andMMAF analogues

ON

OMe

O

R

HN

O

R

HNR

R

NHO

NH

OMe

R

ROH

H2N

R

R

ON

OMe

O

R

H2N

O

R

NR

R

OH

O

R

HN

OHPG

PG OH

HNOtBu

OOMeMe .HCl

NOMe

OH

OBoc

Manufacture of Auristatin Analogues

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Challenges: Chemical stability Non-crystalline Purification

Manufacture: kg scale Larger scale if required

(1000L reactors)

Purification: Precipitation

OH

NH

HN

NH

O

O

NH

NH2O

O

N

O

O

MaleimidoCaproyl p-aminobenzoyl

valine-citrulline

Manufacture and use of linker

FG1 FG2

MaleimidoAmino

Carboxylic acidActivated esterActivated carbonate

AmidesCn chainsPEG chainsVal-Cit

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NHO

NOMeO

NOMe

O

R

HN

O

R

HNR

RR

R

O

NH

HN

NH

HN

R

O

O

O

NH

NH2O

R

O

NO2O

O

NH

HN

NH

HN

R

O

O

O

NH

NH2O

R

ONHO

NOMeO

NOMe

O

R

HN

O

R

NR

RR

R

Linker

DrugLinker

Drug

Linker + drug (cytotoxic payload)

Manufacture 100s of grams scale Larger scale if required Purification Reverse phase Biotage Preparative HPLC

Challenges Non-crystalline Purification

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• Targeted therapies (eg ADCs) is a growing area of interest within the biopharmaceutical industry

• Increased need for radiolabelled biomolecules for A(D)ME evaluation

• Carbon-14 Labelling on Linker and Drug components of the ADC

Summary

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Department of Biocatalysis & Isotope Chemistry

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Thank you

The hexagonal shapes denote the famous Giant’s Causeway rock in Northern Ireland – these shapes also connect to the benzene ring used in science