Week 1 HW: Final Project Ideas

Recombinant Wound-Healing Growth Factor via E. coli

(1) What I want to biomanufacture, and why I want to biomanufacture recombinant human Epidermal Growth Factor (rhEGF), a small (~6.2 kDa, 53-amino-acid) signaling protein that stimulates keratinocyte and fibroblast proliferation and accelerates re-epithelialization of skin wounds. The target output is purified, biologically active rhEGF formulated into a topical cream/gel. My motivation is utilitarian and personal at once: chronic non-healing wounds (especially diabetic foot ulcers) are a major driver of lower-limb amputation worldwide, and topical growth-factor therapy is one of the few treatments shown to meaningfully speed healing. Existing approved products (e.g., rhEGF and rhPDGF-BB gels) are expensive and often unavailable in lower-resource settings. A low-cost, microbially-produced version — something that could plausibly be made with benchtop fermentation and automation rather than a large pharma facility — is interesting to me because it connects a real clinical need to a manufacturing process that is small enough to imagine building myself. It’s for patients with chronic wounds and for clinics that can’t afford imported biologics.

(2) What living system would I use? Chassis: Escherichia coli (e.g., BL21(DE3) with a pET/IPTG-inducible T7 promoter system), because it’s fast-growing, cheap, genetically simple, and the standard first choice for small, disulfide-containing therapeutic proteins at prototype scale. In: glucose/glycerol carbon source, nitrogen source, trace minerals, O₂, IPTG (inducer). Process: Transform E. coli with a plasmid encoding mature hEGF fused to a His-tag (and optionally a periplasmic secretion signal, since EGF has 3 disulfide bonds and needs an oxidizing environment to fold correctly — the cytoplasm alone often produces inclusion bodies). Grow to mid-log phase, induce with IPTG, express for several hours, harvest cells, lyse (or osmotic shock if periplasmic), and purify by affinity chromatography, with a refolding step if the protein comes out as inclusion bodies. Out: purified, folded, bioactive rhEGF; biomass and spent media as waste. Assumption I’m making explicit: disulfide-bond formation is the main risk here — I’m assuming either periplasmic export or use of an engineered cytoplasmic-folding strain (e.g., SHuffle) gets me usable protein, but this needs to be verified experimentally.

(3) How would I use automation? I’d use a desktop liquid-handling robot (Opentrons OT-2) to automate the strain/condition screening step of the workflow — specifically, testing combinations of IPTG concentration, induction temperature, and induction time in 96-well plates to find the condition that maximizes soluble, folded protein rather than inclusion bodies. The same platform would automate small-scale affinity-resin purification in filter plates and set up plate-based assays (BCA protein quantification, and a fibroblast-proliferation bioactivity assay) to score each condition. This matters because folding/solubility, not just expression level, is the real bottleneck for a disulfide-rich protein — automating this design-build-test-learn loop lets me screen dozens of conditions in parallel instead of one flask at a time, which is exactly the kind of high-throughput workflow described in Petzold & Mukhopadhyay (2025) for accelerating biomanufacturing process development.

(4) How much, and how long? (a) How much. Target scale: enough rhEGF to supply a full topical treatment course to 1,000 diabetic-ulcer patients. Assumptions: Formulation: 100 μg rhEGF per gram of cream (0.01% w/w), in line with the order of magnitude used in approved rhEGF/rhPDGF wound gels. Dose: 1 g cream applied twice daily. Treatment course: 8 weeks (56 days), typical for chronic-wound healing trials. Protein per patient = 100 μg/g × 1 g × 2/day × 56 days = 11.2 mg For 1,000 patients = 11.2 mg × 1,000 = 11.2 g finished active protein Assuming ~50% loss through purification/refolding/polishing (a common order-of-magnitude for refolded proteins), I’d need to produce ~22.4 g at the fermentation stage.

(b) How long. Assumption: achievable folded/bioactive titer of 30 mg/L (conservative for a disulfide-rich protein in standard E. coli; crude expression can be much higher, but folding losses dominate). Culture volume needed = 22.4 g ÷ 0.03 g/L ≈ 750 L Prototyping scale: 2–4 weeks of Opentrons-driven DBTL cycles (mL-scale shake flasks/96-well) to lock in expression conditions. Production scale: a single ~750 L–1,000 L fed-batch fermentation run (growth + induction ≈ 2–3 days) followed by downstream purification/refolding/polishing/QC (≈ 5–8 days) → roughly 10–14 days per campaign if run as one large batch; more realistically, using 2–3 smaller (200–400 L) parallel reactors, ~3–4 weeks for the full 1,000-patient batch.

(5) What I don’t know yet Actual folded/bioactive yield — the 30 mg/L titer is a guess; real soluble, correctly disulfide-bonded EGF yield from E. coli needs to be measured (via bioactivity assay, not just total protein). Downstream recovery efficiency — the 50% purification/refolding loss is an assumption; real recovery could be much lower if aggregation dominates, which would multiply the required culture volume. Formulation stability — whether rhEGF stays active and stable in a topical cream over a realistic shelf life is unknown and would need accelerated stability testing.

AI use note (for the syllabus requirement): This draft was generated with Claude (Anthropic), prompted with the assignment PDF plus the topic “Wound-healing Protein / E. coli / Growth factors / therapeutic proteins,” asking it to help with plan and understanding of a back-of-envelope calculation.