ProductionProduced over 100 lots of AAV from transfection to final fill and finish, including characterization assays. Lots ranged from research scale productions to scales for mouse and NHP testing. Upstream DevelopmentDeveloped Suspension HEK293 Transient Transfection AAV production platform, increasing titers 22-fold and decreasing costs Carried out multiple rounds of optimization including Cell Culture Media testing, Reagent Selection, Transfection Plasmid Ratio Improvement, Cell Density testing Downstream DevelopmentWorked with Principal Scientist to develop in-house Tangential Flow Filtration and AKTA Chromatography Improved Benzonase treatment process though buffer reformulation, implemented final sterile filtration of vectors with minimal product loss, testing ultracentrifuge gradient process and capsid distribution in layers. Identified and tested final fill containers that have minimal protein binding. AssaysCarried out first in-house Capsid ELISA testing, developed in-house Alkaline Gel Electrophoresis and Endotoxin testing protocols Frequently utilized ddPCR for Vg Titer assay as well as RT-ddPCR for multiplexed transcriptional analysis assaysCarried out cell-based assays such as vector MOI evaluation via Flow Cytometry In-Vivo Study CharacterizationExtraction of DNA, RNA, and protein from hundreds of mouse tissues as well as NHP tissues.Conducted biodistribution testing of drug product in multiple mouse tissuesDeveloped ddPCR primers and probes for RPP30 housekeeping gene in macaques. Participated in the evaluation of new primers and probes for other gene expression targets. Determined RNA Integrity Number (RIN) scores for mouse and NHP tissues.

Carried out all processes involved in AAV production as part of the Production and Development team producing ~72 lots of 1 E+13 vg AAV per week, >99% success rate. In 2022, helped produced 2098 AAV lots and in 2023 increased to 2425 AAV lots. Upstream: HEK293 Expansion of adherent and suspension cells, Plasmid Transfection using both calcium phosphate and pei processes, , Harvest/cell lysis Downstream: Denarase Incubation, Clarification, PEG precipitation, Ultracentrifugation, Dialysis Maintained production plasmid inventories, sequenced all incoming plasmids to assure identity and purity Prepared hundreds of samples for external sterility testing. Carried out endotoxin testing interpreted and communicated results, maintained recordsDrafted and reviewed process SOPsContributed to 5S implementationPresented a poster at a Sangamo work retreat

As part of the Viral Vector Development team, successfully up-scaled from bench to commercial scale a cGMP biological drug substance production process, 20L harvest scaleProject owner of process development for cell scale up in iCellis Nano adherent bioreactor system (40,000 cm2)Contributed to developing downstream process including UF/DF, Benzonase endonuclease addition, and final product fill and labeling. Trained GMP operators on processes in preparation of multiple successful GMP production runsDrafted and reviewed SOPs for PD grade processesReviewed and edited batch record protocols for cGMP processes as well as IND and BLA enabling paperwork Designed and executed experiments to make data driven decisions for improving vector production processesExtensive cell culture work with HEK293 stable producer cell lines (66,000 cm2 scale)Worked cross-functionally with the Tech Transfer, Analytical Development, Quality Control, and Internal Manufacturing departments to transfer processes into GMP productionSciNote electronic lab notebook (ELN) group administratorPerformed genome titer assay (gc/mL) using Reverse Transcription Digital Droplet Polymerase Chain Reaction (RT-ddPCR)Performed functional transduction unit (TU/mL) assay using flow cytometry

-Carried out human cell culture; both adherent and suspension-Trained and qualified in Flow Cytometry-Performed viral tittering in 96-well plates-Supported viral production method development, transfer, and technical reports as needed-Assisted in writing technical reports, protocols/SOPs, and Tech-Transfer documents-Maintained detailed documentation of laboratory procedures, experiment methods, and data collection

Generated multiple CRISPR protein K/O cell lines Treated cell cultures with inflammatory cytokines and measured cell responses using qrtPCRCultured and maintained mammalian cell lines and primary cells requiring different media formulations at scales up to 4,500 cm2Utilized Western Blot assay to determine successful protein knockout cell linesLab manager duties such as coordinating equipment calibrations, maintaining consumable stocks and scheduling lab meetings. Designed experimental series to determine effects of small molecule anti inflammatory on the growth pattern of renal cancer cells

I helped design and conduct experiments involving human primary PBMCs, three different cancer cell lines, and multiple other primary human fetal and adult tissues. I carried out drug, cytokine, and inorganic molecule treatments on cell mono-layers or in co-culture conditions and would use technical analyses such as FACS, qrtPCR, and automated cell counters. Utilized MACS frequently to isolate cell populations out of PBMCs in bulk to then carry out further differentiation and experimentation. Techniques Practiced:Biosafety Cabinet Sterile ProcedureAseptic Cell Culturing Fluorescence Activated Cell Sorting Magnetic Activated Cell Sortingquantitative reverse transcription PCRDrug TreatmentCell Differentiation