Comparability and Setting Product Specification Limits – A Case Study

Bo Kara, Process Development Advanced Therapy Delivery UKRMP- Comparability: Manufacturing, Characterisation

and Controls Workshop, Cambridge, UK. 14-15 September, 2015

Outline

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1. GSK and Cell Gene Therapy

2. Control Strategy

3. Case Study: • ADA-SCID Gene Therapy

Manufacturing Process and Changes

• Comparability Study Design • Comparability Study Results and

Conclusions

Alliance (2010) GSK- TIGET • Retroviral ex-vivo gene therapy for ADA SCID (lead - optioned 2010)

– Adenosine deaminase (ADA) deficiency, Severe Combined Immunodeficiency (SCID)

• Lentiviral platform for lysosomal storage disorders, primary immune deficiencies and blood disorders: 2 optioned 4Q2013 (MLD, WAS)

– Metachromatic leukodystrophy (MLD), Wiskott-Aldrich Syndrome (WAS)

• Telethon responsible for advancing all programs to PoC

• GSK responsible for global Regulatory, Manufacture and Commercial activity

• Driven by GSK’s commitment to R+D and diversification

Collaboration with Adaptimmune (2014) • TCR engineered T cell immunotherapies for cancer

GSK - Cell and Gene Therapy Platform

(The San Raffaele Telethon Institute for Gene Therapy (TIGET), joint-venture between TIGET and the Telethon Foundation)

“Control Strategy” - Underpins the Product Specification

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“The commercial manufacturing process(es) are controlled by ensuring process parameters (including critical process parameters), in-process controls

and in process specifications are within their expected ranges” • Process Parameter – an independent variable that may be controlled during the execution of an experiment or manufacturing process • Critical Process Parameters (CPP’s) – parameters whose variability may have an impact on a Critical Quality Attribute (CQA) and therefore should be monitored or controlled to ensure the process produces a product of the desired quality • In-Process Controls (IPC’s) – tests/activities performed during production to monitor, and if necessary and appropriate, adjust, the manufacturing process

Deviations form pre-defined parameters or control ranges will be assessed to determine their impact on the relevant CQA(s) prior to batch deposition

• In-Process Specifications – tests performed during production to ensure the product will be of the desired quality

• Product Specifications – (chosen) to confirm the quality of the DS and DP rather than to establish full characterisation:

• molecular and biological characteristics demonstrated to support ensuring the safety and efficacy of the product

Deviations from the in-process/product specifications will trigger and Out of Specification (OOS) investigation

Demonstrating Comparability of Clinical and Commercial Process – Case Study ADA-SCID Autologous Hematopoietic Stem Progenitor Cell (HSPC) Gene Therapy

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Manufacturing Logistics

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Vector Manufacturing

Sterile filtered vector aliquots stored at <-65°C.

CD34+ Cell Separation & Transduction Process

Cell Processing/Manufacturing site (MolMed s.p.a.) co-located with Hospital/Treatment site (San Raffaele

Hospital, Milan, Italy)

Testing and staged release

Bone Marrow Extraction

Patient Infusion

BM Harvest Joint Accreditation Committee - International Society for Cellular Therapy and European Society for Blood and Marrow Transplantation (JACIE). Patients tested : presence of infectious agents as EU Directives 2004/23/EC and 2006/17/EC PP’s, CPPs, IPC’s and

IPS’s

Operating ranges, Set Points and Acceptance

Limits

Why do we need to do anything? • Low volume, acute need - why not just go with it?

• Data shows wide variability, lack of process robustness − e.g. Vector Copy Number (VCN)

• Raw materials, impurities, containment improvements needed

Do something - but not everything... • Changes introduced with the aim to: - Increase scale of manufacture (vector) - Increase process efficiency/performance consistency and robustness - Decrease variability throughout the process - Increase sterility assurance

• ..But no radical redesigning or over-complicating the process

Process Changes

Changes Introduced in Retroviral Vector Process

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End of production cell collection and freezing down

Vector filling and freezing down

Vector filtration

Vector production and harvest

Cell Expansion

MCB bank thaw • Raw materials (replace animal derived raw materials and minimise TSE risk)

• Cell Bank (MCB to WCB)

• Culture conditions (fixed seeding densities, passaging regime, cell factories)

• Optimised vector production (single harvest, temperature, time)

• Scale (~1L/harvest to 12L)

• Additional filtration step (0.45µm to 0.45µm + 0.22µm)

Changes Introduced in Cell Process

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DP manufacture and filling in container closure

DS

Transduction (x3)

Cytokine Pre-stimulation

CD34+ Purification

BM preparation and MNC isolation • Raw materials (minimise TSE risk)

• CD34+ purification (MidiMACS to semi-automated CliniMACS)

• Introduction of range of viral titres used for transduction (titre/VCN correlation, maintain safe and efficacious VCN levels observed in clinic)

• Addition of wash step prior to final DP formulation.

• Introduction of target cell concentration in DP

Comparability Assessment

• Accordance with the principles set out in ICH Q5E

• Process changes minimal: no detrimental effects on product quality anticipated

• Clinical efficacy demonstrated using batches with wide ranges of quality attributes

• For both vector and cell process, comparability assessment performed by:

- Analytical testing and extensive characterisation

- Comparing to clinical data (where possible)

- Commercial ‘equal or better’ than Clinical for comparability batches

- Additional characterisation tests

- where no clinical data was available

- where new assays were introduced for commercial manufacture 10

Comparability Study Design

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2 Full Scale Vector Campaigns 3 Full Scale Vector Batches

2 Vector Campaigns (Study 1)

Transduction 3 lots of pooled BM CD34+ cells

In Vitro comparability Stability

Increased Cell Characterisation

In addition, confirm engraftment for commercial process in murine study

Clinical Process Commercial Process

Study 1 Vector

Study 2 Cells in vitro (HD BM)

* Commercial process runs also used as commercial process validation/verification runs

In Vitro comparability Stability In Vitro

comparability Stability

3 Vector Batches (Study 1)

Transduction 3 lots of pooled BM CD34+ cells

In Vitro comparability Stability

Comparability Results Vector - Potency and Purity

• Viral titre is the main potency test for vector

• Commercial process titres higher than representative clinical but within historical range (and at higher end)

• Acceptance criteria set as ≥ lowest TU/mL used in clinical development with positive outcome

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• Infectivity (titre/total particles) as measure of purity

• No historical data available

• Acceptance criteria set at Commercial ≥ Clinical

✓

Test Acceptance criteria Criteria met?

ADA Activity Activity detected Yes

Presence of Viral RNA (vector/EPC) 197 bp band present Yes

Vector integrity 4.2 ± 0.4 kb Yes

Vector Integration Comparable to MCB reference Yes

Provirus Sequence Confirmation Confirmed against reference Yes

Packaging Components (presence) 692 bp and 567 bp bands present Yes

Packaging Components (relative copy number) Commercial = Clinical* Yes

Comparability results Vector - Identity and Genetic Stability

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*No significant difference in copy number between Commercial and Clinical batches

✓

• Residual BSA: Commercial batches lower than batches 1 and 3 of clinical campaigns:

• Explained by differences in the clinical process operation - multiple harvests, change to serum-containing media between harvests 2 and 3

• HCP: Post-filtration levels of commercial batches higher than clinical • Wash step prior to DP formulation further reduces residuals

• All other tests - negative/in spec (RCR, sterility, endotoxin, mycoplasma, visible particles, bovine viruses, adventitious viruses, clarity)

Comparability results Vector - Residuals and impurities

• No historical data for HCP, host cell DNA, residual BSA

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Test Acceptance criteria Criteria met?

Host Cell DNA Commercial ≤ Clinical Yes

Host Cell Protein (HCP) Commercial ≤ Clinical No

Bovine Serum Albumin (BSA) Commercial ≤ Clinical No

✓

Comparability results Vector - Summary

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• Data obtained supported conclusion - vector manufactured using the clinical process and the commercial process are comparable

- Identity, Integrity and Safety • Changes implemented in commercial process give rise to vector with higher titre

and infectivity - Titre within clinical range • Comparable stability profiles (accelerated, intermediate, long term storage

conditions)

⇒ No impact on any of the vector CQAs

Comparability results Cell Process - %CD34+ in DS

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• Clinical and commercial process batches comparable

• Values within range obtained in the clinic ✓

Comparability Results Cell Process - Clonogenic potential

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• Clinical and commercial process batches comparable

• CFU values higher than historical range - Due to different starting material (healthy donor vs patient bone marrow) - Values within range obtained from process

development experiments (healthy donor)

• Comparable change in clonogenic potential (DS/CD34+) between clinical and commercial batches

• ∆CFU within historical range - Comparability batches and process

development data ✓

Comparability results Cell Process - Vector Copy Number

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• Acceptance criteria based on clinical trials and practical considerations • Clinical process and commercial process deemed comparable for VCN

– Vector distribution within the target cell population assessed qualitatively

✓

Comparability Results Cell Process - Viability

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• Acceptance criteria set to >80% • Clinical and commercial process batches comparable • Results within historical range

Clinical Process Commercial Process

✓

Comparability Results Cell Process - ADA Activity

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• Healthy donor BM with background ADA activity • Results subtracted from background observed in non-transduced samples

• Clinical batches lower than commercial batches (lower VCN)

✓

Comparability Results Cell Process – Additional Characterisation

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• Provirus sequence confirmation • Integration site analysis by LAM-PCR (linear amplification mediated-PCR):

polyclonal appearance for both clinical and commercial batches • Extensive phenotype characterisation using multiple cell surface differentiation

markers - CD45, CD34, CD90, CD133, CD19, CD16/CD56, CD15, CD3

✓

Comparability Results Cell Process - Residuals and Impurities

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• Acceptance criteria set as Commercial ≤ Clinical for all residuals • Residual BSA, Retronectin, cytokines - comparable levels for all batches - Greater clearance from DS to DP for commercial batches due to the wash step • HCP, Host Cell-DNA, free vector particles (total and infectious) - Higher in commercial DS batches (due to higher viral titres used for transduction),

but reduced to <LOQ in DP ✓

Comparability Results Cell Process - Summary

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• DS/DP manufactured using the clinical and the commercial process are comparable

• Higher VCN obtained with commercial process/vector batches - at middle of range obtained in the clinic • Better clearance of residuals and impurities in commercial process DP

⇒No impact on any of the DS/DP CQAs

First to file

• EU MAA for ADA-SCID gene therapy submitted to the EMA (May 2015) • First submission for an autologous HSPC ex-vivo gene therapy

Acknowledgments

TIGET • Alessandro Aiuti, Giuliana Ferrari, Luigi Naldini, Luca Biasco,

Eugenio Montini, Aisha Sauer, Michela Gabaldo, Elena Beltrami

GSK • Nina Kotsopoulou, Aileen Kirkpatrick, Smaragda Angelidou

• Natalie Ward, Christina Basford, Conrad Vink, Tarik Senussi, Sara Nilsson, Sabine Johnson, Celeste Pallant, Natalie Francis, Tom Kaiser, Eirini Vamva

• Jan Thirkettle, Ian Pitfield, Tony Lamus, Renata Lakoma, Ian Blount, Sadia L’Baouch, Michele Myers, Tristan Marshall, Rob Piperno, Alexis Cockroft, Stefano Adami

• Jonathan Appleby, Jim Faulkner, Jason Gardner

• James Walford, David Dow, Nalini Mehta, Victor Neduva, Johnson Yeboah Afari, Alan Lewis, Rhiannon Lowe, Patrizia Cristofori, Jan Klapwijk, Paul McAllister, Damien O’Farrell

MolMed • Paolo Rizzardi, Claudia Benati,

Giuseppina Marano, Francesca Bellintani, Francesca Rosetti, Giuliana Vallanti, Margherita Neri, Alessandra Gatti, Michele Manfredini, Paola Massarielo, Simona Rovetta, Anna Silvani