carbon-14 l abelled adcs dr william h. watters isotope chemistry manager
DESCRIPTION
Carbon-14 L abelled ADCs Dr William H. Watters Isotope Chemistry Manager w [email protected] www.almacgroup.com. Almac overview. Biomarker Discovery & Development. API Services & Chemical Development. Pharmaceutical Development. Clinical Trial Supply. Clinical - PowerPoint PPT PresentationTRANSCRIPT
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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