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Antibody-Drug Conjugates – The Road to the Current State

Nila Das, Ph.D.American Drug Delivery & Formulation Summit

San Diego, CAJune 13, 2016

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Current Status of ADCs

C&E News. 2014, 92(3): 13-21

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Current Status of ADCs (Contd..)and most of them uses one of two classes of drugs ….

C&E News. 2014, 92(3): 13-21

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Mechanism of Action of ADCs

Senter, P.D. and E.L. Sievers. Nat Biotech. 2012, 30(7): 631-637

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Development of an ADC

mAb Conjugation Purification Formulation Shelf-Life

Analytical Measurements

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ADCs are Complex!!

• Each component must be individually characterized and considered for their impact on the ADC as a whole

• Each component contributes to the CQAs of an ADC

Rostami, S. et al. J Antibody Drug Conj. Rev. Aug 01, 2014

Kamath, A.V. & Iyer, S. Biopharm Drug Dispos. 2016, 37(2): 66-74

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Critical Quality Attributes of ADCsBiologic Drug Substance Intermediate

Post Translational ModificationsPrimary SequenceHOSGlycan ProfileBiological ActivityPhysical and Chemical Stability

Drug Substance Intermediate / Drug-Linker

Conjugation ChemistryLinker ChemistryHydrophilic/Lipophilic linkersPotency / ImpuritiesStability

Antibody-Drug Conjugates

Biologic Drug Substance Intermediate CQAsDrug Substance Intermediate CQAsUnconjugated mAbUnconjugated free drugDrug Antibody RatioStability

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Conjugation ProcessNon-specific Lysine Conjugation Interchain Cysteine Conjugation Site Specific Conjugation

Wakankar, A. et al. mAbs. 2011, 3(2): 161–172Shen, B. et al. Nat. Biotech. 2012, 30: 184-191

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Conjugation Process – Cysteine Linked Conjugates

• Positional isomers from cysteine – linked conjugates

• Only non-covalent association is available between the antibody heavy chains (HC) and light chains (LC), due to the disruption of interchain disulfide bonds

Guo, J et al. Bioconjug. Chem. 2016, 27: 604−615

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Conjugation Process• Structural heterogeneity at the molecular level

• Difficulty to optimize biological, physical and pharmacological properties –Robustness of the product

• Challenges due the presence of antibody and drug-linker in a single molecule

• Complex manufacturing process at the conjugation and DS manufacturing level

• Lysine conjugation reaction leads to some bonding with tyrosine forming a less ester bond, which leads to release of free drug over time

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Conjugation Process• Case Study 1 Random conjugation

Accelerated Development

Akash, M.S.H et al. Internat. J. Polym. Mat. and Polym. Biomat. 2016, 65(1): 1-10

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Conjugation Process

The UV spectrophotometric assay developed initially for the DAR measurement was found to overestimate the actual drug loading of the molecule due to interference with a by-product

LC-MS measurement was subsequently employed as the orthogonal method

Lesson: Unreliable data may delay the timing of FIH treatment

Lot DAR by UV DAR by LC-MS 1 3.2 2.3 2 3.3 2.4 3 3.2 2.3

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Payload-Linker Stability• Impact of drug-linker stability

on free unconjugated drug, unconjugated mAb, average DAR and the drug distribution

• Will the unconjugated mAb compete with the ADC?

• Will the free unconjugated drug cause side effects to the patient?

• Will the physical, chemical, pharmacokinetic profiles of the ADC change?

• If the drug-linker stability is poor at the selected solution pH, then lyophilization may be the control strategy !!

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Payload-Linker Stability

Process Flow for Production of T-DM1 from Tmab, via the Key Intermediate T-MCC

Kim, M.T. et al. Bioconjug. Chem. 2014, 25: 1223−1232

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Payload-Linker Stability• Incubation of TMCC showed an

increase in aggregate level after 14 days at 40°C, while T-DM1 and Tmab alone did not show a significant change under the same condition

• What will be the nature of these aggregates?

• CE-SDS confirmed non-disulfide mediated covalent cross-links

• Role of free maleimide on TMCC in forming cross-links with free thiols has been confirmed

• Will the free unconjugated drug form various metabolic products or endogenous long circulating adducts in-vivo?

Wakankar, A et. al. Bioconjug. Chem. 2010, 21: 1588–1595

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Linker

• Peptide linkers such as Phe-Lys or Val-Cit are relatively hydrophobic and can form aggregates, such as in the preparation of doxorubicin conjugates

• Addition of a hydrophilic methoxytriethylene glycol chain via a hydrazone bond greatly inhibited aggregation of the final conjugates

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Payload• Solubility of payload – Often organic solvents may be

required to solubilize payload (e.g. Mylotarg®).

• Impact of such organic solvents on the stability of ADC ?

• If the payload deconjugates, what will be the possible impact on the appearance, stability of the ADC?

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Monoclonal Antibody – Melting Point• Impact of conjugation on mAb

structure and stability

• What will be the impact on the thermal properties of mAbs?

Beckley, N.S. et al. Bioconjug. Chem. 2013, 24: 1674-1683

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Monoclonal Antibody – Aggregation• Many of the small drug

candidates are relatively hydrophobic, and their addition would enhance the hydrophobicity-driven aggregation tendency

• Conjugation via lysine residues would reduce the number of protein surface charges and reduce the hydrophilicity of the protein

• Impact of conjugation process on aggregation, kinetics of aggregation, nature of aggregates

• What will be the impact of increased aggregation tendency on manufacturing, shipping and distribution, and shelf-life of the product?

Guo, J et al. Bioconjug. Chem. 2016, 27: 604−615

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Monoclonal Antibody – Aggregation• Formation of protein

aggregates was a major problem during preparation of antibody- calicheamicin conjugates, and the reaction conditions/additives had to be selected to reduce the tendency of conjugate aggregation

• High ionic strength increases aggregation tendency, more so for ADCs relative mAbs

Yilma, A.T. et. al, Bioconjug. Chem. 2014, 25: 656−664

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Monoclonal Antibody – Aggregation• High payload affects overall

stability of ADCs

• The aggregation tendency becomes more pronounced in high ionic strength formulation with increasing DAR

• Be careful with selection of salts, buffers and their concentrations !!

Yilma, A.T. et al. Bioconjug. Chem. 2014, 25: 656−664

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Product Development of ADCs• Formulation buffer same as the conjugation buffer

• Lyophilization versus liquid (higher cost, time-consuming)

• Nitrogen overlay in vials, minimization of oxygen bubbles, protection from light (minimization of oxidation)

• Stabilizers (sugars, salts tonicity) – Mostly effective to minimize physical aggregation

• Surfactants (Polysorbates) - Prevent surface adsorption and aggregation; peroxide formation

• EDTA, DTPA - Prevent transition metal ion catalyzed oxidation

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Product Development of ADCs• Long Term stability studies - shelf-life statement, dosage form selection

(liquid or lyo)

• Accelerated studies – To support excursions during shipping and in clinics

• Bulk Hold Studies – To define manufacturing conditions

• Compatibility studies – To demonstrate compatibility and stability with the manufacturing train of equipment

• Stress studies – To support excursions

• Photo studies – To define manufacturing conditions, shelf life statement, excursions

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Manufacturing• Facility assessment needs to be performed as early as possible for

extremely potent cytotoxic payloads

• For multi-product facilities handling highly potent substances, a risk assessment is expected to identify cross-contamination risks

• Effective containment technology

• Single use technology

• PE Baseline Guide Volume 7: Risk-Based Manufacture of Pharmaceutical Products: A Guide to Managing Risks Associated with Cross-Contamination 2010

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Clinical

• Will NaCl aggravate on the hydrophobicity and aggregation of ADCs?

• Will the aggregation tendency of ADCs be different from the parent mAb?

• Will the diluent D5W interfere with the stability of the ADC during administration?

• Use-time studies – To define the conditions of clinical administration

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Comparability of ADCs• Depends on whether changes are made to the mAb, payload, linker,

DS or DP

• Extent of comparability will depend on the stage of the asset

• Comparability is needed among toxicology, clinical and commercial batches

• Use as many combinations of distinct drug/ linker/mAb lots as possible during clinical development

• Will the drug distribution remain the same?

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Conclusions…..• ADCs are both small molecules and biologics!!

• The CQAs of the mAb, drug-linker and DS will strongly dictate the quality of the drug product.

• Appropriate measures are needed regarding potential cross-contamination in multi-product facilities.

• Availability of orthogonal analytical methods is critical to characterize and evaluate the physical, chemical, and biological properties of ADCs throughput the product development, as well as, to support comparability

• Communication with Health Authorities is always encouraged !!

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Acknowledgements• Tony Mazzeo• Mark Arnold• Peter Lemaire• Atul Saluja• Many talented members of Bristol-Myers Squibb

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Antibody-Drug Conjugates – The Road to the Current StateCurrent Status of ADCsCurrent Status of ADCs (Contd..)Mechanism of Action of ADCsDevelopment of an ADCADCs are Complex!!Critical Quality Attributes of ADCs Conjugation ProcessSlide Number 9Conjugation ProcessConjugation ProcessConjugation ProcessPayload-Linker StabilityPayload-Linker StabilityPayload-Linker StabilitySlide Number 16PayloadMonoclonal Antibody – Melting PointMonoclonal Antibody – AggregationMonoclonal Antibody – AggregationMonoclonal Antibody – AggregationProduct Development of ADCsProduct Development of ADCsManufacturingClinicalComparability of ADCsConclusions…..AcknowledgementsSlide Number 29Slide Number 30