Week 1 HW: Final Project Ideas

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What you would like to biomanufacture and why?

Target Product: Rapid, on-demand recombinant human desB30-insulin (a fully functional, active 51-amino-acid insulin analog with an intact disulfide architecture missing only ThrB30).

Intended Users: Diabetic patients in low- and middle-income regions, remote clinical sites, or emergency crisis zones facing acute medicine supply chain disruptions.

Motivation & Importance:

  1. Over 96% of the global insulin volume is monopolized by three pharmaceutical conglomerates, resulting in inflated pricing, chronic rationing (affecting 1.3 million Americans alone), and clinic availability dropping as low as 55% in LMICs.
  2. Conventional cell-based biomanufacturing relies on centralized multi-thousand-liter bioreactors and requires weeks to extract, denature, refold, sulfitolyze, and polish insulin from E. coli inclusion bodies.
  3. By producing therapeutic-grade mature insulin locally in under 24 hours, a point-of-care cell-free platform decentralizes biomanufacturing, bypassing cold-chain fragility and single-supplier bottlenecks.

What Living System Would You Use?

  • Chassis Platform: A purified E. coli-derived cell-free protein synthesis (CFPS) chassis (PUREfrex® 2.1) coupled to an on-column enzymatic conversion matrix.

  • System Inputs: Linear or plasmid DNA encoding an engineered proinsulin precursor construct (His-R-IP; ~10.85 kDa). 36 purified E. coli transcription and translation enzymes, ribosomes, tRNAs, amino acids, and NTPs (void of endogenous proteases/peptidases).

    1. Periplasmic folding chaperones: Skp (seventeen-kilodalton protein, 100 µM) and FkpA (peptidyl-prolyl cis/trans isomerase).
    2. Redox shuffling agents: $8\text{ }\mu\text{M}$ E. coli DsbC (disulfide isomerase), $4\text{ mM}$ reduced glutathione (GSH), and $3\text{ mM}$ oxidized glutathione (GSSG).
    3. Downstream reagents: Immobilized Metal Affinity Chromatography (IMAC) Ni-NTA resin and sequencing-grade trypsin.

  • Biological & Biochemical Processes:

    1. Transcription/Translation & Assisted Folding: T7 RNA polymerase transcribes the plasmid, and ribosomal machinery translates the construct. DsbC alongside Skp/FkpA stabilizes nascent hydrophobic regions (e.g., A13–A19, B9–B19) to correctly orchestrate native disulfide bridges ($A7\text{–}B7$, $A20\text{–}B19$, and $A6\text{–}A11$) while preventing irreversible aggregation.
    2. Affinity Capture: The N-terminal $6\times\text{His}$ tag captures soluble proinsulin onto a Ni-NTA matrix without prior cell lysis or inclusion body denaturation.
    3. On-Column Digestion: Reconstituted trypsin cleaves at the engineered Arg13 and the C-peptide flanking dibasic cleavage sites. An engineered K78A mutation eliminates unwanted side-products like Arg-A0-insulin.

  • System Outputs: Mature human desB30-insulin (~5,717 Da) and cleaved by-products ($6\times\text{His}$ leader, C-peptide).

How would you use automation?

  • Prototyping Stage (Bench Screening & Optimization): A desktop liquid-handling robot (e.g., Opentrons OT-2 / Flex) automates micro-scale titration arrays (chaperone stoichiometry, redox ratios, DNA templates, and trypsin digestion times) directly into 96-well plates to maximize active monomer fractions.
  • Point-of-Care Distributed Manufacturing (Automated Execution):
    1. An automated, closed-loop microfluidic synthesis and purification device (such as the Bio-MOD platform).
    2. Workflow Automated:** Automated metering and fluidic mixing of lyophilized cell-free extract with DNA and chaperone reconstitution buffers.
    3. Temperature-controlled incubation ($37^\circ\text{C}$ for 16 hours).
    4. Pressure-driven solid-phase loading across a single-use Ni-NTA affinity column.
    5. Timed perfusion of trypsin digestion buffers (2.5 hours at $37^\circ\text{C}$), followed by automated low-pH quenching and elution.In-line spectrophotometric QA/QC and flow-through sterile filtration.

4. Scale and Production Timeline Calculations

(a) How Much?

  • Defined Clinical Scope: Emergency localized supply for a regional clinic supporting 1,000 insulin-dependent diabetic patients for 30 days.
  • Dosage Assumptions (Borhani et al., 2025):Average daily therapeutic dose per patient $\approx 1.4\text{ mg}$ (equivalent to ~40 IU).

(b) How long: about 20 hours

  • Empirical Process Yields from Literature (Borhani et al., 2025):

    1. Soluble proinsulin CFPS harvest yield (with Skp chaperone) $= 35.1\text{ }\mu\text{g/mL} = 0.0351\text{ g/L}$.
    2. Overall on-column capture and conversion efficiency $= 9.7%$.
    3. Net mature desB30-insulin yield per liter:$$\text{Net Yield} = 0.0351\text{ g/L} \times 0.097 \approx 0.0034\text{ g/L} = 3.4\text{ mg/L}$$

  • Production Time Calculation:

  1. Batch Cycle Time per Run:CFPS Reaction: $16.0\text{ hours}$
  2. Column Capture & Wash: $0.75\text{ hours}$
  3. On-Column Trypsin Digestion: $2.5\text{ hours}$
  4. Elution & CEX Polishing: $0.75\text{ hours}$
  5. Total Cycle Time $\approx \mathbf{20\text{ hours}}$.

What don’t you know yet?

  • Unintended Divalent Cation Chelation on Ni-NTA: Mature desB30-insulin retained unexpected affinity for the Ni-NTA column following cleavage due to endogenous metal-coordinating residues (HisB10, GluB13), demanding an imidazole elution step instead of purely autonomous release. Shifting to an orthogonal capture chemistry (e.g., Strep-Tag II / Strep-Tactin) must be experimentally validated.
  • Co-Chaperone Antagonism and Low Conversion Efficiency (9.7%): Combining Skp and FkpA resulted in lower monomer yields than Skp alone, and significant product was lost to non-covalent proinsulin-insulin heterodimers (~16.7 kDa) and incomplete cleavage intermediates. Optimizing the trypsin-to-protein ratio and introducing β-trypsin variants will require systematic kinetic screening.
  • Oxygenation and Mass Transfer Scaling Limitations: Scaling cell-free reactions above micro-volumes often leads to titer drop-offs due to insufficient surface-area-to-volume ratios and oxygenation constraints necessary for energy cofactor regeneration. Overcoming this requires testing thin-film, bubble-free, or microfluidic bioreactor geometries.

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