Phage-Based Circular ssDNA Manufacturing
Custom cssDNA development and purification through a biology-driven, fermentation-based production platform. From construct design and phage amplification to downstream purification and analytical release, we help nucleic acid therapy developers obtain high-quality circular single-stranded DNA at the scale their program requires.
What Is Circular ssDNA?
Circular single-stranded DNA (cssDNA) is a covalently closed nucleic acid molecule with no free ends. Its topology can offer practical advantages over linear or double-stranded formats for selected gene-editing, expression, and engineering applications.
Covalently Closed Topology
The single-stranded sequence is ligated into a continuous circle, removing the 5' and 3' termini found in linear ssDNA and the double-stranded backbone characteristic of plasmid DNA.
Improved Stability Profile
Because cssDNA lacks free ends, it is less susceptible to exonuclease degradation than linear ssDNA. This can support longer persistence in vitro and in selected in vivo models.
Application Relevance
cssDNA is being explored as a donor template for homology-directed repair, an expression cassette, a building block for DNA nanotechnology, and a reagent for synthetic biology workflows.
Why Phage-Based Production?
Our approach uses filamentous phage biology as an amplification engine. The target ssDNA sequence is carried by an engineered vector, propagated in a suitable host strain, and recovered as cssDNA after controlled disruption and purification.
Biological Amplification
Phage propagation amplifies the target construct during high-density fermentation, providing a biological route to longer ssDNA fragments than solid-phase chemical synthesis can routinely achieve.
Construct Flexibility
The vector backbone is engineered around the sequence of interest, enabling project-specific cassette design and iterative optimization of yield and stability.
Industrial Fermentation
The platform leverages well-established upstream bioprocessing tools, including controlled temperature, pH, dissolved oxygen, and feed strategies, supporting scale-up from shake flasks to 200 L and beyond.
Production Approach Comparison
| Feature | Chemical Synthesis | Enzymatic Production | Phage-Based Platform |
|---|---|---|---|
| Sequence Length | Limited | Moderate | Long fragments |
| Scalability | Low | Moderate | High |
| Cost Efficiency | Low | Moderate | High |
| GMP Readiness | Low | Moderate | High |
| Industrial Manufacturing | Difficult | Challenging | Established |
Reference ranges reflect internal platform development. Actual performance depends on construct length, sequence complexity, and project-specific development.
From Construct to Released cssDNA
An end-to-end bioprocess from phage-mediated biogenesis to high-purity isolation. Each stage is developed and controlled around the specific construct and intended application.
Monoclone Screening
Co-transformation, plate selection, single-colony picking, and glycerol stock preparation. Candidate clones are screened for cssDNA yield and purity.
Clone screeningFermentation
Seed train, tank fermentation (5 L to 200 L), and controlled culture with monitored temperature, pH, DO, agitation, and fed-batch feeding.
UpstreamPhage Harvest
Microfiltration or centrifugation followed by nuclease treatment and TFF, tailored to fermentation scale and phage titer.
HarvestPhage Capture
Anion-exchange capture (e.g., CIMmultus QA monolith) with gradient or isocratic elution to isolate phage particles and reduce process impurities.
CaptureLysis & Clarification
Controlled alkaline disruption of phage particles, neutralization, and depth filtration through graded media to 0.22 µm.
DisruptioncssDNA AEX Capture
Anion-exchange chromatography (Q or DEAE resins) with detergent wash to reduce endotoxin and gradient elution for high-purity cssDNA collection.
PurificationTFF
Tangential-flow filtration for buffer exchange, concentration, and diafiltration into the target formulation buffer.
TFFFill and Finish
Sterile filtration, project-specific aliquoting, vial/tube filling, labeling, and packaging under controlled conditions.
FinishQuality Control & Release Testing
Analytical characterization, batch review, and release assays against agreed quality specifications.
QCDocumentation and Products Delivery
CoA, batch records, regulatory documentation support, and secure shipment of released product.
DeliveryRepresentative Process Performance
mg/L fermentation productivity
phage separation recovery
overall process recovery
cssDNA purity by HPLC
Ranges observed during internal development; not guaranteed for every construct. Acceptance criteria are defined per project.
Capabilities That Support Scale-Up
We develop each unit operation around the construct, impurity profile, and intended use, rather than applying a one-size-fits-all process.
Construct & Host Optimization
Design and cloning of target sequences into expression vectors, host strain evaluation, and clone screening for yield and product quality.
Upstream Process Development
DoE-driven fermentation optimization, scale translation from shake flasks to 5 L, 10 L, 30 L, 100 L, and 200 L bioreactors.
Harvest & Clarification
Centrifugation or microfiltration, nuclease treatment, TFF development, and conditioned media or cell-lysate clarification.
Chromatography Development
Phage capture on CIMmultus QA monoliths, cssDNA AEX capture on Q/DEAE resins, method scouting, and column packing/qualification.
Impurity Reduction
Endotoxin removal strategies, host-cell protein and DNA reduction, residual nuclease control, and nick-opened DNA removal.
Scale-Up & Tech Transfer
Process scale-up, batch consistency evaluation, analytical bridging, and documentation support for downstream manufacturing.
Analytical Characterization
Release and development-stage assays are selected according to the product's intended use, quality target product profile, and regulatory pathway. The table below summarizes methods and reference specifications available on the platform.
| Assay | Method | Reference Specification |
|---|---|---|
| Appearance | Visual inspection / USP <631> | Clear, colorless liquid |
| Identity | AGE / Sequencing | Consistent with theoretical length and sequence |
| UV Purity | A260/A280 ratio | ≥ 1.8 |
| AGE Purity | Agarose gel electrophoresis | ≥ 90% |
| cssDNA Purity | IP-RP-HPLC / UPLC | ≥ 90% |
| Concentration | UV absorbance at 260 nm | ~1 mg/mL |
| Endotoxin | Gel-clot / Dynamic chromogenic / USP <85> | < 20 EU/mg (reference) |
| Host Cell Protein | ELISA | < 1 µg/mg API |
| Host Cell DNA / RNA | qPCR | < 10 µg/mg API / < 1 µg/mg API |
| Residual Nuclease | ELISA | < 1 µg/mg API |
| Microbial Limit | Membrane filtration / USP <71> | < 10 CFU/mL |
Note on endotoxin: internal reference is < 20 EU/mg; fnal acceptance criteria are agreed per project and intended use.
Case Study: GFP-Encoding Circular ssDNA
A representative development project demonstrating the platform's ability to produce functional, high-purity cssDNA from a phage-based process.
Project Overview
Develop a scalable process for circular ssDNA encoding green fluorescent protein (GFP).
Achieve sufficient yield, remove host residuals, and obtain a product that supports functional GFP expression.
Phage-mediated amplification, TFF clarification, and anion-exchange purification.
Takeaway: The result shows that phage-based cssDNA can reach high purity and support downstream functional expression. Actual yields and recoveries remain construct-specific.
Integrated CDMO Support
A multi-purpose facility in Hangzhou with dedicated upstream, downstream, analytical development, and quality control labs supporting the full cssDNA development lifecycle.
Facility
Over 4,000 m² of dedicated labs and GMP suites, including Grade D upstream and Grade C/C+A downstream and fill-finish areas.
Scale
Fermentation systems from shake flasks to 5 L, 10 L, 30 L, 100 L, 200 L, and up to 1,000 L non-GMP, plus cGMP clinical material production up to 200 L.
End-to-End Services
Custom cssDNA development, process optimization, purification development, analytical characterization, scale-up support, and project-specific manufacturing.
Custom cssDNA Development
Construct design, vector assembly, clone screening, and early process definition.
Process Optimization
DoE-based fermentation and purification development focused on yield, purity, and robustness.
Purification Development
Chromatography and TFF process design, resin screening, and impurity clearance studies.
Analytical & QC Release
Identity, purity, concentration, residual, and microbiological testing tailored to release requirements.
Let's Discuss Your cssDNA Manufacturing Requirements
Share your target sequence, intended application, quantity, purity requirements, and development stage.
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