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Carbon-14 L abelled ADCs Dr William H. Watters Isotope Chemistry Manager

Carbon-14 L abelled ADCs Dr William H. Watters Isotope Chemistry Manager w illiam.watters@almacgroup.com www.almacgroup.com. Almac overview. Biomarker Discovery & Development. API Services & Chemical Development. Pharmaceutical Development. Clinical Trial Supply. Clinical

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Carbon-14 L abelled ADCs Dr William H. Watters Isotope Chemistry Manager

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  1. Carbon-14 LabelledADCs DrWilliam H. Watters Isotope Chemistry Manager william.watters@almacgroup.com www.almacgroup.com

  2. Almac overview Biomarker Discovery & Development API Services & Chemical Development Pharmaceutical Development Clinical Trial Supply Clinical Technologies Analytical Services Commercial Services

  3. API services and chemical development

  4. 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 14C radio labelling: API and IMP

  5. Discovery of 14CMartin Kamen & Sam Ruben (27-FEB-1940) T1/2 ~ 5730 Years

  6. [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

  7. Choice of radiolabel • 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.

  8. 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. 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

  10. Strategy: [14C]-Linker Chemistry Drug mAb

  11. Step 1: Drug - [14C]Linker Activation • [14C]-Linker (1 eq) reacted with Protein Drug (viamaleimidelinkage) • IPC analysis by HPLC to determine completion of activation • Reaction temperature critical to minimise degradation • Unbound [14C]-Linker removed using DF (10 kDamembrane)

  12. 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

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

  14. 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

  15. CASE STUDY 2: [14C]-Biomolecule • Specification: • 240 mg of [14C]-biomolecule • Specific Activity > 320 mCi/mmol

  16. Stage 1: [14C]-Peptide

  17. Stage 2: PEGylation

  18. Stage 3: Bio-conjugation

  19. 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).

  20. 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 Manufacture of MonomethylAuristatinbuilding blocks

  21. 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: • Biotage chromatography(kg scale) • Preparative HPLC(15cm column)

  22. Manufacture and use of linker Challenges: • Chemical stability • Non-crystalline • Purification Manufacture: • kg scale • Larger scale if required(1000L reactors) Purification: • Precipitation

  23. Linker + drug (cytotoxic payload) Manufacture • 100s of grams scale • Larger scale if required Purification • Reverse phase Biotage • Preparative HPLC Challenges • Non-crystalline • Purification

  24. 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

  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

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