Molecular Biology, Expression and Upstream CMC

Working with a small team to express (CHO stables/Transients) and characterize (kinetics/binning) Harpoons therapeutics and targets. Scales from 96-well to 5L shake flask. Our Team:*Tittered and normalized (on a Tecan Fluent) 96-well plate expressions for the assay team. *Ran SDS page gels and western blots (as needed) for productions*Conducted early stage PD work for media and feed optimizations and worked with CMO's for later stage PD work.*Carried out Expressed Harpoons molecules as well as antigens and anti-IDs used in QC and or PK assays. *Carried out small scale (24DW filter plate based) purifications to support binning and kinetic studies.*Responsible for ensuring results are captured in Benchling.Developed HT captured kinetic screening for Harpoons molecules on the Carterra LSA (SPR). The method allowed us to generate high quality kinetics and evaluate species cross reactivity as well as specificity from the first 96-well expressions of new binders. The method uses anti-ID binders to capture the same active versions of Harpoon's molecules that are used in functional assays, this meant only one version of the binder needed to be cloned and expressed, reducing the molecular bio and expression work-load.Also developed BLI (Octet) based kinetic assays for non-GMP QC of our clinical molecules.Member of the binders project team (we evaluated cross functional data to select lead binders for new targets) as well as part of the team developing a new therapeutic modality at Harpoon.

•Based on in-house instrument evaluations, provided guidance on IgM’s imaging platform for single round cell-line development and carried out the instrument qualification study for the instrument chosen. •Cloned a cell-line for a Phase 1 campaign at IgM. Identified top performing clones with high titers in shake flask and excellent product quality attributes, i.e. glycosylation state and low aggregation.•First cloning campaign on the platform yielded higher producing IgM clones then a CRO was able to.•Evaluated knock-in and knock-out CHO cell lines to generate clones which produce dramatically improved glycosylation patterns.•Developed a high-throughput cytometry-based titer assay for clone screening.•Characterized IgMs from CHO pools by carrying out a plate-based batch purification, quantified glycosylation states, aggregation (SEC), PQ attributes by ELSIA and product quality on an SDS page gel.•One Associate Scientist as a direct report.

•Evaluated imaging and seeding technologies to reduce CLD efforts to a single round of sub-cloning. Oversaw demos for subcloning cells using microfluid FACS units from Namocell and Nanocellect. Evaluated the Solentim VIPS, and the ALS CellCelector.•Developed subcloning conditions for CHO-S which yield ~70% outgrowth of single cells in a 96-well plate. To do this I arranged an extended demo with ALS Automated Lab Solutions to use their CellCelector which expedited this process by decreasing the outgrowth readout from weeks to days. •Generated stable CHO pools using Atums transposase technology and the Neon transfection system.•Sub-cloned and ran fed batch fermentations with the resulting CHO IgM producing cell lines. Material generated from these campaigns was used in cyno studies. •Sub-cloned, screened, and selected Ramos CDC resistant cells from a pool of CDC surviving cells previously generated at IgM. •Carried out CDC, TDCC (Luciferase or fluorescence-based assays), TCA (T-Cell activation) and FACS based binding assays to characterize IgM antibodies. •Purified IgMs as needed on AKTA's or through small scale batch purifications.•Arranged a demo with Carterra which yielded KD and sandwich binning data for lead hybridoma candidates. Summarized the data relating sequences to binning and KD differences.

*Generated stable cell-lines for immuno-oncology studies.*Sequenced antibodies from hybridoma and evaluated sequences for similarities and redundancy.*Implemented a 3'-race method to simultaneously sequence and clone hybridoma VH and VLs into chimeric (human Fc, mouse Fab) expression vectors.*Carried out kinetic and binning studies on an Octet.*Evaluated antibody TMs and purity using DSF, DLS (Wyatt)

Developed a method for generating a bivalent combinatorial library of two, distinct, flexibly linked ligands. The library consisted of pairwise assembly of low affinity ligands. The bivalent library generated an order of magnitude greater number of high affinity ligands to the model target of lysozyme. The bivalent ligands were shown to express well and retained near wild-type thermal stability. Furthermore, the component ligands comprising a selected high affinity bivalent ligand were unlinked and used in a quantitative mix and read assay for quantifying lysozyme in an impure mixture. The ligands were functionalized by being linked to two of the components of a GFP tripartite system that had been previously developed.In a second project, in the course of making a high affinity ligand targeting beta catenin's C-terminal transactivation domain we discovered we had selected for a disulfide bonded dimeric form of Sso7d. This mutant spontaneously dimerizes after purification from E. Coli and dialysis of reducing agents. Functionality of the dimeric, disulfide bonded form was determined using BLI. The ligand was selected as follows: a monomeric, naive Sso7d library was pre-targeted to the last 12 residues of Sso7d and then selected low affinity ligands were affinity matured through mutagenic PCR to bind full length beta catenin. Derivatives of the dimeric ligand described were the only members in the highest affnity pool of ligands. One clone was studied in detain and was found to have a KD of 5 nM to beta catenin. This is achieved through multi-eptiope engagement of the ligand to both the C-terminus and ARM domain of beta catenin. Interestingly, multi-epitope engagement is also observed for most natural beta catenin C-terminal binding partners.

Evaluated process alternatives for converting and recovering a dilute acetate fermentation product. Worked with research assistants to collect kinetic and phase equilibrium data at the bench scale as well as at a gallon per day pilot scale. Organized and summarized data for in house meetings to review process options and to put process flow sheets together for process cost estimation.Achievements: Co-inventor of a novel base recycle process. Designed and built a pilot unit to generate scale-up data and evaluate operability. Major contributor to development of Zeachem's ethanol recovery process.

Contributed to a Six Sigma Green Belt Project to reduce the batch time of a crystalization process step by studying batch to batch variations to identify the best process control inputs. Realized a 5% reduction in batch time, allowed other process optimizations downstream to realize increased increased production.Managed a $600,000 facility upgrade project that reduced risks of LOPC's by installing corrosion resistant materials and a system to add and track the concentration of corrosion inhibitors. The project also decreased process downtime during maintenance by adding storage and piping connections.

Contributed to the development of a heterogeneous catalysis process capable of decoupling two key plant products. Identified a catalyst by running and gathering data from a high throughput catalyst screening mini-plant with an on-line GC and mass spec. Developed a kinetic model and regeneration process upon which the design was a based. Collaborated with process engineers on a design, capital cost, and determined an NPV.

Scaled the production of photoactive polymer 10X from bench to 200 liter pilot scale. Process documentation also met GMP standards as the piloting was done in a facility normally used for the development of GMP processes.

Created a database that characterized the process conditions of several Saran Recipes that were currently in production. Made statistical process control charts for use by plan operators to monitor the process with. Generated a report correlating production data to computer simulations and research data that was available.