carbon-14 l abelled adcs dr william h. watters isotope chemistry manager

26
1 Carbon-14 Labelled ADCs Dr William H. Watters Isotope Chemistry Manager [email protected] www.almacgroup.com

Upload: kynan

Post on 22-Feb-2016

30 views

Category:

Documents


0 download

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 Presentation

TRANSCRIPT

Page 1: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

1

Carbon-14 Labelled ADCs Dr William H. Watters

Isotope Chemistry Manager

[email protected]

Page 2: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

2

Almac overview

BiomarkerDiscovery &Development

API Services& Chemical

Development

PharmaceuticalDevelopment

ClinicalTechnologies

ClinicalTrial Supply

AnalyticalServices

CommercialServices

Page 3: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

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

Page 4: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

4

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

Page 5: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

Discovery of 14CMartin Kamen & Sam Ruben (27-FEB-1940)

T1/2 ~ 5730 Years

Page 6: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

[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

Page 7: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

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.

Page 8: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

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

Page 9: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

9

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)

Page 10: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

10

Strategy: [14C]-Linker Chemistry

Drug

mAb

Page 11: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

11

• [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

Page 12: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

12

• [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

Page 13: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

13

• [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

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

14

• 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

Page 15: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

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

Page 16: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

16

Stage 1: [14C]-Peptide

14CGly

AA-SEQUENCE LINKER

AA-SEQUENCE LINKER

Boc

H2N Resin

14CGly

Boc COUPLING

14CGly

AA-SEQUENCE LINKERBoc

CLEAVAGE

Resin

Page 17: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

17

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

Page 18: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

18

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

Page 19: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

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).

Page 20: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

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

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

Page 21: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

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)

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

Page 22: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

22

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

Page 23: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

23

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

Page 24: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

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

Page 25: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

25

Department of Biocatalysis & Isotope Chemistry

Page 26: Carbon-14  L abelled ADCs  Dr William H. Watters Isotope Chemistry Manager

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