1

Carbon-14 Labelled ADCs

Dr William H. WattersIsotope Chemistry Manager

william.watters@almacgroup.com

www.almacgroup.com

2

Almac overview

BiomarkerDiscovery &Development

API Services& Chemical

Development

PharmaceuticalDevelopment

ClinicalTechnologies

ClinicalTrial Supply

AnalyticalServices

CommercialServices

3

1 Small molecule development

2 Biocatalysis + Isotopic Labelling

3 Peptide and protein technology

4 Physical sciences

5 Analytical services

API services and chemical development

4

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 radiolabel

Radioisotope Half Life14C 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 terms

Common units used in Radiolabelling

MilliCurie (mCi), and microCurie (Ci) for quantity

Alternative Units

Megabecuerels (Mbq) (1mCi = 37Mbq)

Specific Activity

Commonly expressed in mCi/mmol or Ci/mg

Labelling one carbon atom with 14C results in a maximum

specific activity of 62.4mCi/mmol

•

9

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)

10

Strategy: [14C]-Linker Chemistry

Drug

mAb

11

• [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. Ultrafiltration

Protein-linkerconjugate

14C

14C1.

Step 1: Drug - [14C]Linker Activation

12

• [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

13

• [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

14

• 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

14C

GlyAA-SEQUENCE LINKER PEG

mAb

CASE STUDY 2: [14C]-Biomolecule

16

Stage 1: [14C]-Peptide

14C

GlyAA-SEQUENCE LINKER

AA-SEQUENCE LINKER

Boc

H2N Resin

14C

GlyBoc COUPLING

14C

GlyAA-SEQUENCE LINKERBoc

CLEAVAGE

Resin

17

N

O

O

14C

GlyAA-SEQUENCE LINKER PEG

14C

GlyAA-SEQUENCE LINKERBoc

N

O

O

PEGN

O

O

O

Boc

N

O

O

14C

GlyAA-SEQUENCE LINKER PEG

Boc - Deprotection

Stage 2: PEGylation

18

N

O

O

S

14C

GlyAA-SEQUENCE LINKER PEG

mAb

N

O

O

14C

GlyAA-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

14C

GlyAA-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).

20

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

O

N

OMeO

N

OMe

OHN

O

HN

MMAE

NH

O

OH

O

BnO.HNcy2

HNOtBu

OOMeMe

Z-Ile-OH.DCHA

.HCl

N

OMe

OH

OBoc

N

OHBoc

Boc-Prolinol

NH

HO

O

N

OMeO

N

OMe

OHN

O

HN

MMAE

21

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)

NH

O

N

OMeO

N

OMe

O

R

HN

O

R

HN

R

R

R

R

MMAE andMMAF analogues

O

N

OMe

O

R

HN

O

R

HN

R

R

NH

O

NH

OMe

R

ROH

H2N

R

R

O

N

OMe

O

R

H2N

O

R

N

R

R

OH

O

R

HN

OHPG

PG OH

HNOtBu

OOMeMe .HCl

N

OMe

OH

OBoc

Manufacture of Auristatin Analogues

22

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

FG1FG2

MaleimidoAmino

Carboxylic acidActivated esterActivated carbonate

AmidesCn chainsPEG chainsVal-Cit

23

NH

O

N

OMeO

N

OMe

O

R

HN

O

R

HN

R

R

R

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

O

NH

O

N

OMeO

N

OMe

O

R

HN

O

R

N

R

R

R

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

24

• 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

25

Department of Biocatalysis & Isotope Chemistry

26

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