From Cesium to X-Ray: A Cell Therapy Perspective Emily Hopewell, PhD, MT(ASCP) Moffitt Cancer Center Cell Therapy Facility

Objectives Why irradiators are used in cellular therapy products Why we moved to X-ray How can we demonstrate comparability between the two sources?

Moffitt Cancer Center Cell Therapy Facility Hybrid clinical (BMT) and shared resource for cellular clinical trials Average 425 BMT transplants annually 9 active investigator sponsored IND protocols Manufacturing of cellular products for industry partners Involved in KITE CAR-T trials and SOC, preparing for Novartis CAR-T

Why and what do we irradiate? Irradiate cells to inhibit replication but maintain desired characteristics What? Irradiate feeder cells used in cellular therapy cultures Irradiate tumor cell lines used in vaccine production

Process Development Technology Transfer Scale up and out Validation Moffitt has two cesium irradiators (BB and vivarium), some researchers prefer cesium.

Transition from Cs to X-ray Moved to new facility in 2013 Now what? Always used Blood Bank Cesium irradiator Cells have to be transported from clean room facility to blood bank irradiator - what do we do with other products that are in the middle of a trial? Purchased our own irradiator How do we validate a new irradiator

FDA Recommendations – Blood Components Process validation for blood component irradiation Measure amount of irradiation absorbed by product Use dosimetry system (with dose map) Periodically check for radiation leakage (Geiger counter) For non-gene modified products 2000 FDA Guidance for Industry: Gamma Irradiation of Blood and Blood Products: A Pilot Program for Licensing

Dose Rates and Mapping of X-RAD 160 Maximum: 2.982 cGy/min Minimum: 0.697 cGy/min Average: 2.763 cGy/min % Variation: 76.63%

Standardizing positioning to minimize variation

Minimizing Variation Maximum: 2.982 cGy/min Minimum: 0.697 cGy/min Average: 2.763 cGy/min % Variation: 76.63% Maximum: 2.982 cGy/min Minimum: 2.775 cGy/min Average: 2.908 cGy/min % Variation: 6.94%

FDA Recommendations – Gene Modified Products Provide data to demonstrate cells are: replication-incompetent maintain desired characteristics Describe documentation of calibration of the cell irradiator source 2008 FDA Guidance for Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs)

Verification of radiation of a clinical cell line Compare the effects of X-RAD 160 and Gammacell 3000 on modified K562 line used in trials Endpoints for release testing for trial: Compare release testing results using the two sources of irradiation Characteristic Test Method Acceptance criteria Viability/Cell Count Trypan blue exclusion Decrease over time CD40L Expression Flow cytometry > isotype/negative control GM-CSF Secretion ELISA > 170 ng/1x106 cells/24hr

Cell Characteristics Viable cell numbers declined in both irradiated conditions. There was no statistically significant difference in the viability between X-Ray and Gamma radiation for all time points (p=0.73, two-tailed t-test). Mean ± SD. CD40L expression was not significantly different when comparing radiation source. (p=0.928, two-tailed t-test) Mean + SD. Secretion of GM-CSF showed no statistically significant difference in CD40L expression when comparing radiation sources. (p=0.779, two-tailed t-test) Mean + SD

Comparison Conclusions Successfully met required release criteria regardless of radiation source No statistically significant difference between the two sources Power of the statistics is limited because performed tests three times Subsequent lots produced using X-Ray radiation also met release criteria as expected

Summary Side-by-side comparisons required for production changes during trial Standardization of irradiation technique minimizes product to product variation Positioning Volume restrictions Utilize process development period to ensure: Replication deficient Desired characteristics maintained

Thank you! Moffitt Cell Therapy Facility Linda Kelley, PhD Martha Hackett, MPH Cheryl Cox, MT Ludivina Lay, MD, MT Ana Landin, PhD Moffitt Blood Bank Kaaron Benson, MD Susan Cook, MT, SBB Tamara Busby, MT