1. 1 Carbon-14 Labelled ADCs Dr William H. Watters Isotope Chemistry Manager william.watters@almacgroup.com www.almacgroup.com

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

3. 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. 4 14 C 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

5. Discovery of 14 C Martin Kamen & Sam Ruben (27-FEB-1940) T 1/2 ~ 5730 Years

6. [ 14 C]-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 RadioisotopeHalf Life 14 C5730 years 3H3H12.3 years 35 S87.6 days 125 I60.1 days 131 I8 days 32 P14.3 days 33 P25.3 days Almost all pharmaceutical studies with small molecules are done with 14 C. 14 C present in the skeleton of all drug molecules. 14 C is Detectable at very low concentrations (scintillation counting) Long half life means no need for correction for radioactive decay. 3 H is also used but is more subject to exchange.

8. 14 C 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 14 C results in a maximum specific activity of 62.4mCi/mmol

9. 9 Specification: [ 14 C]-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: [ 14 C]-mAb-Protein Conjugate

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

11. 11 [ 14 C]-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 [ 14 C]-Linker removed using DF (10 kDa membrane) Step 1: Drug - [ 14 C]Linker Activation

12. 12 [ 14 C]-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 Step 2: Antibody Conjugation

13. 13 [ 14 C]-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 Step 3: Purification / Formulation

14. 14 4.36 g [ 14 C]- mAb-Protein Conjugate obtained 21% Radiochemical yield from [ 14 C]-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

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

16. 16 Stage 1: [ 14 C]-Peptide

17. 17 Stage 2: PEGylation

18. 18 Stage 3: Bio-conjugation

19. Summary: Case Study 2 250 mg of [ 14 C]-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. 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

21. 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) Manufacture of Auristatin Analogues

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

23. 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. 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. 25 Department of Biocatalysis & Isotope Chemistry

26. 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