Pharmaceutical Development for ADCs

Presenters

Lisa Hardwick Wendy Saffell-Clemmer

Antibody Drug Conjugates (ADCs)

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!  Consist of: –  a Monoclonal Antibody –  Chemical linker and –  Cytotoxic

!  Monoclonal antibody –  Typically targets tumor-associated antigens on the surface of cancer cells –  Effectiveness requires internalization of the ADC in the cell

Antibody Drug Conjugates (ADCs) !  Linker cleavable vs. non-cleavable –  Cleavable linkers rely on internal cellular processes to release the cytotoxin –  Non-cleavable linkers require degradation of the conjugate !  Cytotoxin – must be highly potent –  Calicheamicin –  Duocarmycins –  Auristatins –  Myotansinoids Image from http://www.adcetris.com

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Current ADC Platforms

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ADC – Surge in INDs

Source- S. Miksinski, presented at AAPS NBC, May 23, 2012

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What Makes an Optimal ADC?

Source: Current Cancer Drug Targets, 2009, 9, 982-1004

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What Makes an Optimal ADC Drug Product Formulation? !   Stability of Linkage

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!   Stability of Mab –  Aggregation –  Fragmentation –  Deamidation Because of stability concerns related to the Mab, all ADCs we have worked with have been lyo products.

Liquid Formulations •  Advantages of Liquid Formulation –  Ease of use –  Less expensive manufacturing

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•  Disadvantages of Liquid Formulation –  Usually more unstable formulation –  Drug product is often frozen at -80°C –  Complicated cold chain management

Lyo Formulations •  Advantages of Lyo Formulation –  Higher probability of technical success –  Better stability –  Frozen storage not required.

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•  Disadvantages of Lyo Formulation –  More complicated development –  More expensive manufacturing –  Specialized capabilities required

The Pharmaceutical Development Process

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!  Timing: Clients come to us for formulation development in two different scenarios: –  Preclinical –  Manufacturing scale-up optimization (Phase I +) !  Clients often desire an aqueous solution formulation instead of lyo but this is not always possible !  Common to pursue dual path where the goal is to develop drug product formulation suitable for either a sterile solution or a freeze-dried solid presentation. –  The formulation is designed with lyophilization in mind, meaning that lyo-suitable buffers and stabilizers are selected.

The Pharmaceutical Development Process

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!  The Dual-Path Process –  Establish Analytical Methods –  Study the Effect of pH, Buffer, Ionic Strength and Surfactants on the Chemical and Physical Stability of the ADC –  Biophysical Characterization by FTIR and Calorimetry –  Screening of Candidate Formulations under Stressed Conditions –  Long Term Stability of Selected Formulations •  Stability studies solution formulation and freeze-dried solid will be carried out concurrently. –  If a freeze-dried formulation is required, carry out additional studies to optimize the process

The Pharmaceutical Development Process

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!  The Dual-Path Process: Lyo Optimization –  Characterize the formulation by low temperature thermal analysis and FD Microscopy –  Develop the Design Space for Primary Drying • Verify appropriate conditions through laboratory runs –  Optimize Secondary Drying Conditions • Verify the effect of residual moisture on stability through short term accelerated stability testing using multiple moisture levels.

Preliminary Solution Stability Studies for the Mab, & the Mab + conjugated drug

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!  Examine factors that influence solution stability –  pH (usually examine range of 5 to 8) –  Buffer (Phosphate, Histidine, Citrate, Tris) –  Polysorbate !  Examine Physical –  Ionic strength Stability –  Protein concentration –  Visual !  We use stressed stability –  HIAC or Micro Flow conditions Imaging (MFI) –  40°C for up to two weeks –  Size Exclusion –  Freeze-thaw Chromatography (SEC) –  Agitation –  Photostabilty

Thermal Characterization

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!  FTIR of the Amide Region –  Evaluate in multiple formulations –  Use as a reference spectrum for examination of formulation and processing effects !  DSC –  Determine if changes in solution composition effect the Tm Midpoint temperatures for the formulations containing 30, 45, and 60 mg/ml sucrose are approximately -31o, -33o, and -32oC, respectively.

Accelerated Lyo Stability Testing !  Evaluate multiple formulations using a conservative lyophilization cycle

!  Place both solution and lyo formulation screening samples on accelerated stability –  Typically use 40-50°C –  Monitor using stability indicating methods

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Accelerated Lyo Stability Testing !  Major changes observed on stability –  Aggregation • Visual • MFI or HIAC • SEC • Dynamic Light Scattering (DLS) –  Charge Heterogeneity • Imaging Capillary Electrophoresis (iCE)

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Long Term Stability

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!  Place liquid and/or lyo formulations on long term stability with full panel of analytical methods

• 24 months • 6 months • 24 months • 3 months

Common Analytical Methods for ADCs

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Stability Indicating Analytical Methods

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!  While methods on the previous page are a standard toolbox, not all ADCs behave the same in all methods –  In Particular, iCE must be optimized for every molecule –  Drug Antibody Ratio –  Free-Drug !  In our experience, the most stability indicating methods have been –  SEC –  iCE

Free-Drug – Stressed Sample Results Lyophilized Sample – 2 Weeks @ 50°C (Red) Lyophilized Sample – T0 (Blue) Free Drug RS @ 0.5 µg/mL (Black) Resolution Solution (Pink) Quenched Drug Linker RS

No Free-Drug detected during long term stability Free Drug RS @ 0.5 µg/mL

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Drug Linker RS

DAR-HIC: Stress Comparison

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DAR 4

DAR 4

DAR 2 DAR 6 DAR 0

DAR 2

DAR 6

DAR 8

Lyophilized Sample – T0

DAR 0

Solution Sample – 2 Weeks @ 40°C

DAR 8

SDS-PAGE – Mab vs ADC

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HHLL HHL HH HL

H

L

Molecular Weight Marker & Monoclonal Antibody (Nonreduced)

Molecular Weight Marker & Monoclonal Antibody (Reduced)

Molecular Weight Marker & ADC (NonReduced)

Molecular Weight Marker & ADC (Reduced)

No changes were observed in SDS-PAGE on stability

SEC ADC Stressed Comparison

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Short Term Stability - Liquid

Aggregate Aggregate

Fragment

Solution: T0,

Solution: 1 week

Post-filtration

@ 50°C

ICE Comparison of two different ADCs iCE e-gram of ADC 1

iCE e-gram of ADC 2

!   Different Linkers produce dissimilar charge isoform profiles

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ICE Stressed Comparison iCE e-gram at initial time point:

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iCE e-gram of the same sample liquid formulation after 1 week at 50⁰C:

High temperatures appear to cause substantial deamidation

Lyo Cycle Optimization !  If the choice is made to proceed with the drug as a lyophilized product, the cycle is optimized for maximum efficacy and efficiency –  Primary drying will occur at the shelf temperature and chamber pressure that will allow for the fastest suitable sublimation of ice from the product. –  The transition to secondary drying will occur at the maximum rate suitable for the product, and the duration will be timed to achieve the optimal residual water content of the finished product

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Thermal Characterization !   Freeze-Dry Microscopy –  Determine Tc of ADC –  Evaluate Tc in trial formulations

-32 C

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FDM of an ADC Formulation Collapse Temperature -30C

-30 C

-27 C

DSC for Thermal Transitions in Frozen Systems

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Lyo Cycle Optimization – Primary Dry

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Design Space !  Measure the vial heat transfer coefficient and the resistance of the dried product layer to flow of water vapor –  This information allows us, by using well established equations for heat and mass transfer in vial freeze drying, to calculate relationship between the controlled variables (shelf temperature and chamber pressure) and the product temperature. !  This forms part of the design space for primary drying, with the other boundary established by equipment capability –  We use tunable diode laser absorption spectroscopy (TDLAS) as an in-process mass flow meter to measure the rate of sublimation, thus enabling these measurements.

Lyo Cycle Optimization – Primary dry

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Design Space

Carry out 1-2 experimental runs to verify appropriate primary drying conditions Monitor cycles by means of product thermocouples (minimum of three), comparative pressure measurement, and by TDLAS (tunable diode laser absorption spectroscopy).

Lyo Cycle Optimization – Secondary Dry Determine Influence of Residual Moisture on Stability of the Drug Product !  Carry out 1-2 experimental runs to measure the rate of secondary drying and establish an appropriate end point for the cycle. –  Monitor secondary drying rate by taking thief samples for moisture analysis using at least two secondary drying shelf temperatures. –  Use Karl Fisher titration for analysis of residual moisture. NIR can be used in addition

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Lyo Cycle Optimization – Secondary Dry !  An additional trial cycle can be run to generate samples for stability testing –  Use a sampler to remove vials at various points during secondary drying. Use near IR for non-destructive measurement of residual moisture !  Monitor physical and chemical stability during stability testing under stressed conditions !  Use these data to support a residual moisture specification

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Residual Moisture - Why Do We Care? !  It’s particularly important for freeze-dried protein formulations –  In general, the stability of freeze-dried proteins is a more sensitive function of residual moisture than small molecules –  Drier may not be better in terms of stability !  Examining the role of residual moisture level on stability is a too-often overlooked aspect of the development plan

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General Approaches

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!  Equilibration at controlled relative humidity –  Usually involves equilibration of freeze-dried product in a desiccator containing a saturated solution of a salt. –  Equilibration times can be rather long. –  Does have the advantage of uniform vial-to-vial residual moisture level. –  The number of options for salts providing a relative humidity of less than about ten percent is rather limited. –  Considerable trial-and-error work is needed to establish the appropriate residual moisture levels, and this can be time consuming. –  Some argue that “back-hydration” is not representative of the real process, although we find no compelling reason to believe this.

Thief Sampling – Our Preferred Method

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Outline of Approach

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!  Carry out a trial freeze dry cycle where, starting at the end of primary drying, a thief sampler is used to remove samples during a series of shelf temperature “steps” in secondary drying. !  A near IR (NIR) instrument is useful for nondestructive measurement of residual moisture, but a reference method is needed, such as Karl Fischer titration. Establish a calibration curve. !  Decide on the number of residual moisture levels to be used, and choose secondary drying conditions accordingly for a second trial run, where thief samples removed for the stability study

Outline of Approach (continued)

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!  Measure residual moisture level of the “thief” samples by NIR, so that, for every sample on stability, there is a reliable estimate of residual moisture in that vial. !  Measure the glass transition temperature (Tg) of the freeze-dried solid as a function of residual moisture, if possible. This is a good indicator of physical stability of the drug product. !  We typically carry out stressed stability testing at 40 and 50C. !  Having an estimate of residual moisture of each vial on stability allows a relationship to be established between residual moisture level and rate of loss of integrity.

Case Study - Cycle with “Step-wise” Secondary Drying AMV159645 REC159646

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Pirani vs. Capacitance Manometer Detail

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AMV159645 REC159646

Water Content as 2° Dry Progresses AMV159645 REC159646

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Residual H2O Analysis

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Karl Fischer

NIR

NIR Spectra

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Calibration of NIR Method

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Sampling During Secondary Drying

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AMV159645 REC159646 5 4 3 2 1

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Residual Moisture for Thief Samples

AMV159645 REC159646

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Representative DSC Thermogram (ADC Formulation with ~3.7% Water)

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AMV159645 REC159646

Tg vs. Residual Moisture

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AMV159645 REC159646

Size Exclusion Chromatography (SEC) AMV159645 REC159646

Monomer

Aggregate Fragment

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Various Levels of Collapse During Stressed Stability Testing at 50oC

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AMV159645 REC159646

Water Content vs. Tg During Secondary Drying

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AMV159645 REC159646 0.3% Tg=75C

1.9% Tg=57C 3.1% Tg=45C 4.8% Tg= 30C

1.0% Tg=66C

0.2% Tg=76C

Stressed Stability Data (Collapsed Samples)

Tg = 38C

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Tg = 47C

Tg = 50C

Postulated Mechanism of the Role of Residual Water Freeze dried solid [Tg – associated mobility]

Collapse Crystallization of Sucrose

Aggregation/Fragmentation

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Note:

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!  It is important to distinguish between collapse as a purely cosmetic defect and a critical defect that could result in sub-therapeutic dosing.

!  In this case collapse promotes the crystallization of sucrose, which causes loss of efficacy as a protectant.

In Conclusion !  ADC Development is a complex process !  Formulation Development –  Advantages to parallel path – liquid and lyo –  Standard approach for protein formulation development appears to be suitable for ADCs –  In our experience so far, stability of the Mab appears to be more of an issue than stability of the linker. • iCE • SEC • No changes observed in Free-Drug by RP-HPLC • No changes observed in Drug Antibody Ratio (DAR) –  No issues with lyo stability up to 18 months

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For Lyo Products !  Design space for primary drying can determine most efficient conditions for both product and equipment !  Comparative pressure measurements can help to determine length of primary and secondary drying !  Residual Moisture can impact stability –  Optimal final water content for protein formulations can differ, although usually higher than for small molecules

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Thank you! !   For information about Baxter’s BioPharma Solutions services, please visit our website at www.baxterbiopharmasolutions.com

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Disclaimer

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This presentation is for informational purposes only, and is provided “as is” with no warranties, express or implied. Although this presentation is designed to provide accurate and authoritative information with regard to the subject matter covered, neither the author nor Baxter accepts responsibility for errors and omissions. This presentation is protected by copyright laws. Individuals may reproduce and distribute this presentation for individual, non-commercial use. All other uses require advance written permission from the author.

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