Carbon-14 Labelled ADCs
Dr William H. Watters
Isotope Chemistry Manager
william.watters@almacgroup.com
www.almacgroup.com
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Almac overview
Biomarker
Discovery &
Development
2
API Services
& Chemical
Development
Pharmaceutical
Development
Clinical
Trial Supply
Clinical
Technologies
Analytical
Services
Commercial
Services
API services and chemical development
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1
Small molecule development
2
Biocatalysis + Isotopic Labelling
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Peptide and protein technology
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Physical sciences
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Analytical services
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
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Discovery of
14C
Martin 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 Life
14C
5730 years
3H
12.3 years
35S
87.6 days
125I
60.1 days
131I
8 days
32P
14.3 days
33P
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
CASE STUDY 1:
[14C]-mAb-Protein Conjugate
Specification:
• [14C]-mAb-Protein Conjugate required carbon-14 label on the linker
• Specific Activity of ≥ 1.1 Ci/mg and 4 g of material
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Strategy: [14C]-Linker Chemistry
Drug
mAb
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Step 1: Drug - [14C]Linker Activation
• [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)
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Step 2: Antibody Conjugation
• [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
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Step 3: Purification / Formulation
• [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
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Summary: Case Study 1
• 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
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CASE STUDY 2: [14C]-Biomolecule
Specification:
• 240 mg of [14C]-biomolecule
• Specific Activity > 320 mCi/mmol
Stage 1: [14C]-Peptide
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Stage 2: PEGylation
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Stage 3: Bio-conjugation
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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
S.L. Kitson, T.S. Moody, D.J. Quinn, A. Hay, ‘Carbon-14 Bioconjugation: Peptides and
Antibody-Drug Conjugates’, Pharmaceutical Sciences, Manufacturing & Marketplace Report, May 8 (2013).
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
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Manufacture of Auristatin Analogues
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:
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
Biotage chromatography
(kg scale)

Preparative HPLC
(15cm column)
Manufacture and use of linker
Challenges:

Chemical stability

Non-crystalline

Purification
Manufacture:

kg scale

Larger scale if required
(1000L reactors)
Purification:

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Precipitation
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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Summary
• 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
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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
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