allan davidson world adc san diego september...

Piramal Method Development Observations and Approaches Allan Davidson World ADC San Diego September 2017

• Discuss Piramal approach for ADC Cell Based Assay method development

via short case study

• Present considerations when developing methods and characterising ADC

/ Bioconjugate products

Objectives

Piramal Method Development Observations and Approaches

Piramal Enterprises Ltd*

Critical Care Consumer Products Decision Resources Group

Piramal Imaging

FINANCIAL SERVICES

• Rated best among real estate fund managers in India & among real estate debt fund managers in Asia

• Contract research & manufacturing

• Presence across North America, Europe & ROW

• Inhalation and Injectable Anesthetics

• Global top 3 by market share in inhalation anesthetics

• India focused OTC business

• Among top 7 OTC companies in India

• Gold standard service provider of healthcare data analytics & insights

• Emerging leader in molecular imaging

• State of art facility in Berlin

• Social initiatives in the field of healthcare, skill development & education

• Pharma and cosmetic glass

• One of the leading global

players in cosmetic glass

• Incorporated in 2007 with

29 m sqft under development

• Major investors include

Warburg Pincus & Goldman Sachs

*Publicly traded entity of Piramal Group

HEALTHCARE

Pharma Solutions

PIRAMAL FOUNDATION

(PHILANTHROPY)

PIRAMAL GLASS LTD. PIRAMAL REALTY LTD.

Piramal Group

3


Piramal Pharma Solutions Global Footprint - Capabilities

Note: * Dietary Ingredients

API & Formulations Development & Manufacturing

USFDA, MHRA

ADC Development & Manufacturing

USFDA, MHRA

GRANGEMOUTH

MORPETH

AURORA

API Development & Manufacturing

USFDA, MHRA

LEXINGTON

SHANGHAI

Sourcing Office

API & Formulations Development

MUMBAI

MAHAD*

Vitamins & Minerals Premixes

USFDA, WHO-GMP

AHMEDABAD

PITHAMPUR

Formulations Manufacturing

USFDA, MPA Sweden

DIGWAL


USFDA, MHRA

ENNORE


WHO-GMP

RIVERVIEW

HPAPI Development & Manufacturing

USFDA

Drug Discovery & Formulation Development

MPA Sweden

4

ADC Sterile Development & Manufacturing

USFDA


Changes in the ADC / Bioconjugation landscape

Piramal Method Development Observations and Approaches 5

• Shift in projects and enquiries

• Increase in small /medium Biopharma

• Push for more development activities

• Much more variety in toxin + linkers

Case Study – Development of a Cell Based Assay and analysis of Herceptin-MMAE conjugate and fractions


• To present our approach for developing cell based assays for Early Phase projects

• Analysis of fractions collected from DAR species

• Considerations for obtaining material to allow characterisation of species

• Consideration to species characterisation in other tests

• Use of the cell based assay in safety / cleaning activities

Scope



Development of a Cell Based Assay to target HER2 expression

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• Assay may be performed in 2 formats, continuous cell culture or with frozen cells. Development work presented is using frozen cells

• Prior to any assay development, optimal cell growth characteristics are established

• Cell morphology and population doubling time are monitored and assessed prior to banking



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• A sub-master cell bank and working cell bank are established

• Each bank is differentiated by 1-2 passages, working cell banks may be differentiated by more than 2 passages

• Based on growth characteristics of the cell line, it may take 3-6 weeks to create a bank of sufficient size to support development, qualification and testing for >1 year

In this case study, MDA MB 361 (ECACC 92020423) classified as 2+ for Her2 expression



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Goal of assay development was to

• Generate an optimised dose dilution series

• Establish appropriate drug incubation time and cell recovery time

• Assess metabolic reagent(s) and incubation time

• Test for specificity and mock potency samples (50-200%)

Not considered for this case study: cell density and full performance characteristics assessment


Development of a Cell Based Assay (Early Phase)

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Data not presented that would typically be assessed for Early Phase • Cell density – Typically 3000 Cells/Well • Cell Recovery time – 1 – 4 Hours or 16 – 24 Hours • Cell Bank robustness – Master cell bank and working cell bank will be assessed for

similarity in performance

• Mock potency samples assessed over 5 point (50, 71 ,100 ,141 ,200 %) • Mock potency samples tested to generate repeatability, I.P, Bias/Accuracy, Linearity

• Specificity also performed


Development of a Cell Based Assay (Late Phase)

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• DoE driven Robustness experiments

• Based around key parameters, eg Operator, Cell bank, Cell number, Drug exposure time, Cell recovery time

• Key Read outs – Potency , SST criteria (EC50 )

• Also considered: Format Variability, Method Capability (Cpm) & OOS Potential per USP <1033>

Where to start?

13 Piramal Method Development Observations and Approaches

• Working cell bank already created

• Start with a 23 point dilution series over anticipated potency range

• Use cell density of 3000 cells/well

• Work with a 3 day drug incubation as we know this is typical duration for MMAE

• Have sufficient plates / rows to perform readouts after both 4h and overnight in Alamar Blue and with CTG

Dose dilution series optimisation and assay readout

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• 23 point dose dilution series

• Day 3 Termination

• Alamar blue readout (4h)

• S/N not optimal


Dose dilution series optimisation and assay readout

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• Alamar blue readout (O/N)

• S/N good


Cell Titer Glo Readout

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• Cell Titer Glo

• S/N >5


Cell Titer Glo versus Alamar Blue

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Alamar Blue Cell Titer Glo


Next Assays

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• 3 day readout suitable but confirm 1 day vs 2 day and 3 day

• Confirm cell recovery time (4h vs overnight)

• Now use 23 point dilution series used to help construct an 11 point dilution series

Assay Termination – Day 1 vs 2 vs 3

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Cell Recovery (Over night vs Same Day)

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No Difference observed irrespective of the day of Drug addition (Over night Recovery vs 4 Hours)

Dose dilution optimisation

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• Established preferred Drug Incubation

• Established suitable metabolic reagent and incubation time

• Confirmed cell recovery time • Two dilution schemes assessed by varying dilution factors. Dilution scheme can

be geometric or may be asymmetric (different factors used around asymptotes to better balance the series)

• Assess assay over 50-200% range (mock potency samples)


3 Day Incubation, Alamar Blue

Preparation of conjugate material


Herceptin Stock

Buffer Exchange

[P]

Modification

Range [TCEP]

90min Ambient

Conjugation

Excess [vcMMAE]

Buffer Exchange

(Formulation)

Mini conjugations

Fraction Collection and Characterisation


Sample selected based on which DAR species being targeted. Multiple species collected per injection Fractions then concentrated using Amicon centrifugal filter units on day of generation Fractions then buffer exchanged into formulation buffer and stored at -20°C Fractions pooled for use in cell based assay Pooled fractions analysed by HIC, PLRP, UV initially

Fraction Collection and Characterisation


HIC Fraction Found DAR

DAR 0 0.00

DAR 2 1.95

DAR 4 3.95

DAR 6 6.12

DAR 8 8.00

PLRP

Fraction Found DAR

DAR 0 0.00

DAR 2 1.81

DAR 4 3.95

DAR 6 6.07

DAR 8 7.97

• Good correlation for DAR • Protein Concentration determined for all pooled fractions (not presented) • What are the potencies of the fractions?


Comparison of Average DAR versus Fraction collected DAR species

• What are the differences in potency between fraction collected drug load species and material with average DAR (4.4)?

• Tested on 3 Plate model with varied sample positions

Following Fractions tested: • DAR 2,4,6,8

• Relative potencies generated

compared to material with average DAR = 4.4



Species Result (%)

DAR 2 37.2

DAR 4 75.5

DAR 6 115

DAR 8 105

Control (100%)

97.1



Same experiments with ADCs generated over range of DAR typically show a correlation and higher potency Size Exclusion therefore performed and clear that significant aggregation had occurred on the higher DAR species

Species Result (%)

DAR 2 93.5

DAR 4 92.8

DAR 6 81.3

DAR 8 61.4



Considerations for fraction collection • Exposure to solvent more of an issue to higher DAR material?

• Collection into a volume of Formulation buffer to compensate prior to buffer

exchange?

• Is formulation optimal if there is a need to store material longer term?

• Highlights the fact that higher DAR material is much more prone to aggregation and to what cost?


Other considerations for product stability / inactivation

• Stability indicating nature of the Cell Based Assay

• Drug inactivation to support health and safety or cleaning activities


Stability Considerations?

• Under stability testing, the potency of the ADC generally drops, but there is also the potential for it to increase, particularly if residual drug-linker or cleaved drug-linker forms more toxic species.

• May well be important that these species can be detected and quantified through other means (RP-HPLC). Presents challenges where drug species have no chromophore but drug-linker does.

See next slide

Non Specific Cytotoxicity Assessment

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• Free Drug or Free Drug Species all are capable of inducing Non specific Cytotoxicity.

• MMAE is much more potent than other species


What other uses does the Cell Based Assay have ?

• Allows generation of data to support drug inactivation / cleaning activities.

• Inactivation studies typically performed with cleaning agents such as NaOH, acid(s) or oxidisers at ranges of concentrations.

• Degradation can be monitored by RP-HPLC with PDA and MS detection to support species characterisation but doesn’t tell us whether the degraded species are cytotoxic or not. Best example is degradation of drug-linker at linker end returning more toxic species…

See next slide


Drug Inactivation

Increase in potency observed relative to drug at equivalent concentrations Data can be generated in parallel by RP-HPLC (MS)

Summary

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• Shown that early phase Cell Killing Assays can be developed quickly and effectively

• In a CMO environment their use goes beyond just release and stability testing. Useful in understanding the inactivation for safety considerations

• Fraction collected material (particularly high DAR) may be much more susceptible to aggregation

• Consider methodology for obtaining fractions, selection of buffer and storage

• Really obvious, but some care should be spent ensuring DAR method is well characterised ! Same could apply to other methods


Thanks to Kranthi Vunnam Analytical Development Robert Wilson PhD Analytical Development Neil Thomson Analytical Development Ian Hannah PhD Analytical Development David Williams PhD Process Development

Thank You


allan davidson world adc san diego september...

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