1 carbon-14 labelled adcs dr william h. watters isotope chemistry manager...
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Carbon-14 Labelled ADCs
Dr William H. WattersIsotope Chemistry Manager
www.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 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
•
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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. Ultrafiltration
Protein-linkerconjugate
14C
14C1.
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
14C
GlyAA-SEQUENCE LINKER PEG
mAb
CASE STUDY 2: [14C]-Biomolecule
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Stage 1: [14C]-Peptide
14C
GlyAA-SEQUENCE LINKER
AA-SEQUENCE LINKER
Boc
H2N Resin
14C
GlyBoc COUPLING
14C
GlyAA-SEQUENCE LINKERBoc
CLEAVAGE
Resin
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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
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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).
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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
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
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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)
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
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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
FG1FG2
MaleimidoAmino
Carboxylic acidActivated esterActivated carbonate
AmidesCn chainsPEG chainsVal-Cit
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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
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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