CQAs for C&GT Products to Enable Comparability Assessment. Ben Thompson Snr Director, Biopharmaceutical CMC RA GlaxoSmithKline

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1 CQAs for C&GT Products to Enable Comparability Assessment Ben Thompson Snr Director, Biopharmaceutical CMC RA GlaxoSmithKline

2 Overview Demonstrate the value of defining CQAs early in product development for Managing process changes and defining formal in vitro comparability studies Managing analytical changes and maintaining comparability of analytical results Describe key learnings and challenges experienced in CMC development of ex vivo gene therapy product Disclaimer: Scenarios presented are hypothetical for illustration only.

3 Critical Quality Attributes CQAs are: -Product attributes with potential to impact safety or efficacy - The foundation for managing product quality through all stages of the product lifecycle CQAs are NOT: - Analytical methods -Specifications A CQA is a physical, chemical, biological or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality. CQAs are generally associated with the drug substance, excipients, intermediates (in-process materials) and drug product (ICQ8).

4 Product Profile Ex vivo Gene Therapy genetically modified autologous CD34 + cells Target: metabolic disorder Data Clinical Data 20 patients treated Follow-up to 6 years (mean = 3 yr) No treatment-related SAEs and clear signs of efficacy in majority of patients In vitro characterisation data 20 batches of product for patient treatment 5 batches of product generated from healthy donors In vivo biodistribution study Compare transduced with mock-transduced cells in NSG mice no difference noted inter-animal variation in engraftment & VCN

5 Step 1: Define the CQAs Identity Potency / Purity Safety Process Related Impurity Cell Product CQAs Percent CD34+ Vector copy number CD34+ Stem Cell Potential Enzyme Activity Cell Viability (%) Transduction efficiency Vector copy number (VCN) Endotoxin Mycoplasma Microbiological Control RC Adventitious virus ost Cell Protein Plasmid DNA ost Cell DNA Residual infectious particles Residual cytokines Vector CQAs Infectious viral titer Potency Infectivity Transgene sequence Identity Vector Integrity Purity Vector infectivity ost Cell Protein BSA Process related impurity ost Cell DNA Benzonase Microbiological Control mycoplasma endotoxin Safety Adventitious virus Plasmid DNA RC

6 Step 2: Identify Analytical Methods Cell Product CQAs Percent CD34+ Vector copy number CD34+ Stem Cell Potential Enzyme Activity Cell Viability (%) Endotoxin Mycoplasma Microbiological Control CP Analytical Method Flow cytometry qpcr assay Clonogenic capacity assay Enzyme assay using PC to detect activity Trypan blue A qpcr BAC T Alert EISA

7 Step 3: Assess Robustness of Analytical Method Ensure consistency of assay performance

8 Impact of Assay Robustness Transduction Efficiency (%) Need for 0 retain samples Batch Need for robust assays early in development Assay 1 Assay 2

9 Proposed Changes Manufacturing Process Component Process v 1.0 Proposed Process v 2.0 Rationale for Change Vector Process Case 1 Cell Process Case 2 Cell expansion Adherent Suspension Enable treatment of larger Cell manipulation Final product formulation Manual production Fresh product with 4 hour shelf life Implementation of automation Cryopreserved product. population of patients including some older patients Improve supply chain robustness

10 Assess Impact of Vector Process Change Vector CQAs Potential Impact Cell Product CQAs Potential Impact Infectious viral titer Percent CD34+ Infectivity Vector copy number Transgene sequence CD34+ Stem Cell Potential Vector Integrity Enzyme Activity CP Cell Viability (%) C DNA Transduction efficiency Benzonase Endotoxin Microbiological Control Mycoplasma mycoplasma Microbiological Control endotoxin RC Adventitious virus Adventitious virus Plasmid DNA M CP RC Plasmid DNA M Capture rationale for outputs to be studied ost Cell DNA Residual cytokines

11 Comparability Study Design Clinical Process Commercial Process 3 full scale vector batches 3 full scale vector batches VS Stability Testing Stability Testing Vector characterisation based on impact assessment 3 Vector batches (Study 1) 3 Vector batches (Study 1) Transduction 3 lots of D apheresis VS Transduction 3 lots of D apheresis Stability Testing Stability Testing Cell characterisation based on impact assessment

12 Define the Model System Use of healthy donor apheresis Prospectively assess impact of use of healthy donor material No expected difference between healthy donor and patient material for safety or impurity attributes. CQAs Vector copy number Transduction Efficiency CD34+ Cell Growth Cell viability Percent CD34+ Clonogenic potential Sequence Enzyme activity Impact of D as Surrogate for Patient Cells No expected difference No expected difference ower growth rate in patient samples compared to D cells No expected difference No expected difference igher clonogenic potential expected in healthy donor No expected difference Expected normal levels in healthy donor material compared to patient cells

13 Assess Impact of Cell Process Change Cell Product CQAs Potential Impact of automation Potential Impact of cryopreservation Percent CD34+ Vector copy number M CD34+ Stem Cell Potential Enzyme Activity M Cell Viability (%) M Transduction efficiency Endotoxin Mycoplasma Microbiological control RC Adventitious virus CP Plasmid DNA ost Cell DNA Residual cytokines

14 Comparability Study Design Ensure sufficient starting material Resources required to run processes in parallel

15 Conclusions Early definition of CQAs is critical for supporting both process and analytical changes Strong analytics can form the bedrock of comparability Comparability exercises need to consider impact of vector, DS and DP changes Model systems for cell-based products are useful tool, and it is critical to understand the relevance of the model