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

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

3. 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

4. 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

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

6. 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

7. 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

8. 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%

9. Standardizing positioning to minimize variation

10. 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%

11. 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)

12. 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

13. 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

14. 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

15. 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

16. 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