Plasmid DNA

Plasmid is propagated not in a test tube but inside E. coli. So the starting point is to prepare, as the seed, a single bacterium 'carrying the right blueprint correctly.'

The seed culture is dispensed uniformly and frozen so that quality is the same in every lot. This is the cell bank, and for each manufacturing run, bacteria are revived from here and grown.

What matters is reading the full-length sequence of the plasmid before storage to fix identity. If the foundation wavers here, however much the later steps are polished, the blueprint itself is off.

Plasmid is propagated inside E. coli. The seed bacteria are frozen uniformly so that every lot can be grown at the same quality. Fixing at the start that 'the right bacteria carry the plasmid with the right sequence' is the starting point of the whole process.

Alternatives in practice

• NGS full-length sequence confirmation · Illumina MiSeq (v2/v3 reagent kits) or Oxford Nanopore MinION (whole-plasmid consensus) — Strong at comprehensive detection of mutations and rearrangements, and outsourcing to services such as Plasmidsaurus is also possible. Avoids the read-length limit of Sanger
• Restriction enzyme fingerprinting · Agilent 4200/5200 Fragment Analyzer or Bio-Rad agarose gel + GelDoc Go imager — Simple identity confirmation by matching the cleavage patterns of multiple enzymes. No sequence information is obtained
• Host 16S rRNA identification/phenotype · bioMérieux VITEK 2 or Bruker MALDI Biotyper (MALDI-TOF MS) — Prevents mix-ups of the host strain (DH5α/DH10B, etc.). Confirms the identity of the bacterial cells, not of the plasmid

The central step where plasmid is actually 'mass-produced.' The seed culture is first expanded in flasks and then moved to the main culture in a culture vessel called a jar fermenter.

The fed-batch method (adding nutrients little by little) is used here. By topping up rather than giving everything at once, the cells are grown to high density while the amount of plasmid per 1 cell is also preserved.

In other words, it is a step that balances 'the number of cells' and 'the share per 1 cell,' and the cell mass and culture conditions here almost entirely decide the final yield.

The seed culture is expanded in shake flasks and then grown in the main culture in a jar fermenter. In the fed-batch mode, nutrients are added little by little to grow the cells to high density, maximizing overall yield while also preserving the amount of plasmid per 1 cell (copy number). The cell mass and culture conditions here determine the final yield.

Alternatives in practice

• Batch culture · Sartorius Biostat B / Eppendorf BioFlo 320 (benchtop to mid-scale) — Simple, but copy number and yield are inferior to fed-batch. For early development
• Antibiotic-free selection (operator repression/auxotrophic complementation) · Host/vector of the Cobra Biologics ORT (Operator-Repressor Titration) system or the Nature Technology RNA-OUT system — When avoiding antibiotic residue in a GMP drug product. A dedicated host strain is required
• Temperature-shift-induced copy number amplification · Eppendorf BioFlo 320 (30→42 °C shift control) — Temperature-sensitive copy number amplification derived from the pUC-type ori. Medium-independent, but heat stress management is needed

The liquid after culture is in a state where the cells are thinly spread, and it cannot go on as is to the step that breaks them. First only the cells are collected and concentrated, and unneeded medium components are washed off.

Collection is by centrifugation or microfiltration. The collected clump of cells is evenly redissolved (resuspended) in the dedicated buffer to be used for the next lysis, completing the preparation.

Unglamorous but needing attention is this redissolving. If lumps remain and it is uneven, the next alkaline lysis works poorly and plasmid recovery falls.

The broth after fed-batch culture cannot be sent directly to alkaline lysis; the cells are concentrated by centrifugation or microfiltration (MF-TFF) to remove medium components and then uniformly resuspended in lysis buffer. If resuspension is incomplete, lysis efficiency and plasmid recovery drop.

Alternatives in practice

• Batch centrifugal pelleting · Beckman Coulter Avanti JXN-26 / J-26S (4,500–6,000 ×g) — Simple at small to mid scale. Unsuited to continuous processing
• Tangential flow microfiltration (concentration and washing) · Repligen KrosFlo hollow fiber (0.2/0.45 µm) or Cytiva ReadyToProcess hollow fiber ULTA — Gentle shear; retains the cells while washing out the medium. Flux management is the key

This is where the shells of the bacteria are first broken and the plasmid inside is taken out. The cells are dissolved with alkali (NaOH and SDS) and the plasmid is released into the liquid.

Neutralizing next with potassium acetate causes the huge genomic DNA, protein and SDS to clump and sink all at once. Only the small circular plasmid remains in the supernatant.

This rough sorting is the key to crude purification. But if too strong, it even breaks the plasmid's 'twist (supercoil),' so how hard it is applied determines quality.

The cells are collected and broken with alkali (NaOH/SDS) to let the plasmid dissolve out. Neutralizing next with potassium acetate causes genomic DNA, protein and SDS to clump and sink, leaving only the small circular plasmid in the supernatant. This step is the key to crude separation, and if overdone, the supercoils are broken.

Alternatives in practice

• Thermal flocculation + solid-liquid separation · Plate heat exchanger + Alfa Laval centrifuge or depth filter — An alternative to chemical lysis that uses no SDS. Suited to automation at large scale
• Batch alkaline lysis + centrifugation · Stirred tank + Beckman Coulter Avanti centrifuge — For small scale and development. Watch for fragmentation of genomic DNA by shear
• RNase-free process design · RNA removal is ensured by downstream AEX/HIC (CIMmultus DEAE, etc.) — When GMP policy is to avoid animal-derived/foreign enzymes. Downstream load increases

On neutralization, genomic DNA, protein and fragments of bacteria float up as a white, cotton-like aggregate. This step removes it to give a clear solution.

The lower layer containing the plasmid is put through depth filtration (straining through a thick filter) and finally sterile filtered at 0.2 µm. The turbidity is eliminated and a clear load solution is prepared.

The aim is to protect the downstream chromatography column. If cloudy liquid is run through, the column clogs, so cleaning up here is a prerequisite for the whole separation.

The white aggregate formed by neutralization (genomic DNA, protein, debris) is removed to give a clear solution. The lower layer containing the plasmid is clarified by depth filtration and the like so that the downstream chromatography column does not clog. Because of the high-salt conditions, adsorptive retention is unlikely to occur, so yield is easy to maintain.

Alternatives in practice

• Disc-stack centrifugation · Alfa Laval Culturefuge 100 / GEA clarifying centrifuge — Effective for primary removal of high solids. Fine debris requires downstream filtration in combination
• Tangential flow microfiltration · Repligen KrosFlo hollow fiber (0.45/0.65 µm) — Continuous clarification of lysate. Membrane fouling management is the key
• Flotation + mesh collection · Dedicated flotation tank configuration — Flotation recovery of SDS precipitate. Reduces centrifuge load for large-scale lysate

The clarified liquid is 'dilute and large in volume.' Loading it as is onto the purification column is inefficient, so first water is removed by ultrafiltration (UF/DF) to concentrate it.

The membrane acts as a mesh that does not pass the large plasmid and passes only salts and small molecules. Using this, the buffer is replaced with one whose composition, such as salt concentration, suits chromatography.

It is, so to speak, conditioning before the main event. With concentration and composition set, more can be loaded at once on the next capture chromatography, and the separation also tends to be stable.

The clarified liquid is dilute and large in volume, so it is concentrated by ultrafiltration (TFF) and the salt concentration and so on are exchanged into a buffer suited to chromatography. Plasmid molecules are large, so they are retained on the membrane while salts and small molecules are removed. It is pretreatment that raises the loading amount and robustness of the next capture chromatography.

Alternatives in practice

• Selective salt precipitation (isopropanol/PEG) · Stirred tank + Beckman centrifuge or depth filter — Combines concentration and crude purification. Solvent recovery and waste treatment are challenges
• Hollow fiber TFF (large pore size) · Repligen KrosFlo hollow fiber (500 kDa to 0.05 µm) — Reduces shear on large plasmids. Balance between flux and genomic DNA permeation
• Concentration by bind-elute chromatography · Sartorius CIMmultus DEAE monolith — Concentrates by capture without going through TFF. Buffer exchange is achieved at the same time

In a process that does not degrade RNA with an enzyme (RNase-independent), the clarified liquid still contains large amounts of high-molecular-weight RNA, genomic DNA and endotoxin.

So calcium chloride or high salt is used to selectively sink and drop these troublemakers. It also serves as preliminary handling that conditions to a salt concentration and conductivity suited to the subsequent anion exchange.

Why is it needed? If these are loaded in excess onto the next column, both the capacity to adsorb and the resolution break down. It is an unglamorous but effective step that sets the loading conditions.

In an RNase-independent process, if the clarified lysate is applied directly to anion exchange, excess high-molecular-weight RNA, genomic DNA and endotoxin break down the column capacity and resolution. Removing these by selective precipitation with CaCl2 or high salt and conditioning to a salt concentration and conductivity suited to the subsequent AEX capture is a standard step.

Alternatives in practice

• RNA and gDNA removal by sequential PEG/CaCl2 precipitation · Stirred tank + Beckman centrifuge / depth filter — Selective precipitation of high-molecular-weight nucleic acids. Trade-off between recovery and purity
• Pre-reduction of endotoxin by Triton X-114 phase separation · Temperature-controlled stirred tank (cloud point separation) — Reduces LPS load before AEX. A step to remove residual detergent is needed

Plasmid has a strongly negative charge on its backbone. So it is adsorbed like a magnet onto a positively charged support (anion exchange), capturing the product all at once.

After capture, it is eluted by raising the salt concentration. In this single step, the major impurities such as host-derived protein, RNA and genomic DNA are all greatly stripped away together.

Plasmid molecules are large, so monolith or membrane type supports that let them reach the interior are preferred. It is a core step that performs capture, concentration and impurity removal at the same time.

Plasmid is strongly negatively charged, so it is captured by strongly adsorbing it to a positive support (anion exchange) and eluting by raising the salt concentration. This single step greatly removes the major impurities such as host-derived protein, RNA and genomic DNA, and concentrates and captures the product. Monoliths and membranes that large plasmids can easily enter are used.

Alternatives in practice

• Bead-based AEX resin (large pore) · Cytiva Capto DEAE / Capto Q ImpRes、Tosoh TOYOPEARL GigaCap Q-650M — High capacity and low cost, but the dynamic binding capacity is rather low because plasmid is limited in diffusion into the pores
• SEC as the primary separation · Cytiva Sepharose 6 Fast Flow / Sephacryl S-1000 SF — Separation is possible without adding salt, but throughput and concentration are limited
• Membrane AEX · Pall Mustang Q (0.8 µm pores)/Sartorius Sartobind Q — High flow rate and disposable, with high binding capacity for large nucleic acids. Lower resolution than resin

Even after capture, unwound open circular and cut linear plasmids still remain, along with endotoxin, which causes fever. This is the finishing step that removes them.

The difference in 'reluctance to mix with water (hydrophobicity)' is used. Adsorbing onto the support at high salt concentration and eluting while lowering the salt separates and removes these impure forms.

The aim is to concentrate at high purity only the twisted form (supercoiled form) desirable for therapy. It is a core step that contributes to meeting the supercoiled fraction specification.

A finishing step that removes the open circular and linear plasmids that remain after capture, and endotoxin. When adsorbed onto a hydrophobic support under high salt, these impure isoforms and endotoxin can be separated, and the supercoiled form desirable for therapy can be concentrated at high purity. A core step that contributes to meeting the supercoiled fraction specification.

Alternatives in practice

• Endotoxin removal by Triton X-114/X-100 phase separation · Temperature-controlled stirred tank (cloud point separation) — Removes LPS efficiently. Management of residual detergent is needed
• Supercoil-selective polishing by thiophilic aromatic adsorption · Cytiva PlasmidSelect Xtra (28-4024-02) — Separates sc from oc/linear with high resolution. Uses high-salt ammonium sulfate conditions
• Polishing by a second-stage AEX · Sartorius CIMmultus QA monolith / Cytiva Capto Q ImpRes — An alternative when HIC is to be avoided. Isoform resolving power is inferior to HIC/thiophilic

The final polishing of purification. Sorting only by differences in molecular size, it separates open circular, linear and multimeric forms and small fragments that the earlier steps could not completely remove.

Size exclusion chromatography is used. Using the property that larger molecules elute earlier and smaller ones later, the supercoiled form desirable for therapy is raised to 90% or more.

The advantage is that desalting and buffer exchange can be done at the same time. It is the standard closing step of plasmid purification that finishes purity while bridging to the next formulation.

Open circular, linear, multimeric and low-molecular-weight fragments that anion exchange and HIC cannot completely remove are separated by size exclusion chromatography to achieve the supercoiled content of 90% or more required by regulation. It also serves for desalting and buffer exchange at the same time, and is the standard final polishing step of pDNA purification.

Alternatives in practice

• Size-exclusion-like polishing with monolith supports · Sartorius CIM/CIMmultus monolith — Avoids diffusion limitation for large plasmids and allows high flow rates. The separation mechanism differs from pure SEC
• Gel filtration polishing with Sephacryl S-1000 SF · Cytiva Sephacryl S-1000 SF + AxiChrom / XK columns — Wide fractionation range for very large nucleic acids. Advantageous for plasmids larger than those suited to Sepharose 6 FF

A step that readies the fully purified plasmid in a form suited to storage and administration. UF/DF replaces it into a buffer such as PBS or TE and matches it exactly to the specified concentration.

Finally it is sterile filtered at 0.2 µm to cut off microorganisms and aseptically filled into vials. The final 'form' as drug substance/drug product is decided here.

Solution stability and sterility are also settled at this stage. Because it becomes the form that reaches patients as an injectable nucleic acid therapeutic, it is an important step that determines how well the formulation turns out.

The purified plasmid is replaced into a buffer suited to storage (such as PBS or TE) and adjusted to the specified concentration. Finally it is sterile filtered at 0.2 µm and aseptically filled into vials. Solution stability and sterility are decided here, and the form as drug substance/drug product is established.

Alternatives in practice

• Lyophilization (naked DNA formulation) · SP Scientific LyoStar / IMA Edwards freeze dryers — Improves room-temperature stability. Requires a lyoprotectant and process development
• Lipid nanoparticle (LNP) encapsulation · Precision NanoSystems NanoAssemblr (microfluidic) — Formulation for in vivo delivery. Control of particle size and encapsulation efficiency is the key
• Frozen bulk storage (−20/−80 °C) · Sartorius Celsius FFT / Cytiva bio-containers + ultra-low temperature freezers — Simplest and stable. Cold-chain management during transport and thawing is needed

Plasmid has 3 forms: the twisted form (supercoiled), the unwound open circular form and the cut linear form. The supercoiled form is what works therapeutically.

The proportion (%SC) is counted accurately by capillary gel electrophoresis, an electrophoresis method using a thin tube. It is a test that cleanly separates the 3 forms and gives the ratio.

Why is it the most important? Because the supercoiled fraction is directly tied to efficacy and ease of entry into cells. The FDA has also recommended specifying roughly 80% or more as a guide.

Plasmid has 3 forms: supercoiled, open circular and linear, and the supercoiled form is desirable for therapy. Its proportion (%SC) affects potency and transfection efficiency, so it is measured as the most important CQA. The FDA has recommended specifying it with roughly 80% or more as a guide.

Alternatives in practice

• Neutral agarose gel electrophoresis + densitometry · Bio-Rad PowerPac+GelDoc Go, Image Lab analysis — Simple and low-cost, but resolution and quantitative accuracy are inferior to CGE
• AEX-HPLC · Agilent 1290 Infinity II Bio LC + Thermo Fisher DNAPac PA200 column — Quantifies isoforms by HPLC. Easy to share equipment
• Alkaline agarose gel electrophoresis (AAGE) · Bio-Rad submarine electrophoresis system — Useful for detecting single-strand breaks (nicks). Complementary to neutral gels

A test that confirms every time, lot by lot, whether the plasmid to be shipped has 'the correct sequence and structure as designed.' It assures there are no mix-ups or mutations.

It is a 2-stage approach. The band pattern after restriction enzyme digestion shows the rough structure, and sequencing checks the sequence letter by letter, confirming agreement with the design.

For therapeutic nucleic acids, this identity is the foundation. If the contents differ, efficacy and safety both collapse from the ground up, so confirmation for every lot is indispensable.

Confirm for every lot that the plasmid to be shipped has 'the correct sequence and structure as designed.' The band pattern after restriction enzyme digestion and sequence verification by sequencing show that there are no mix-ups or mutations. Identity assurance that is the foundation of therapeutic nucleic acids.

Alternatives in practice

• NGS full-length sequence confirmation · Illumina MiSeq or Oxford Nanopore MinION — Confirms the whole-plasmid sequence with high reliability. Also detects minor mutations and rearrangements
• Restriction enzyme fingerprinting alone · Agilent 5200 Fragment Analyzer / Bio-Rad agarose gel — Rapid, low-cost identity confirmation. Cannot assure at the sequence level
• Confirmation of target sequence by PCR/qPCR · Thermo Fisher QuantStudio 5 / Applied Biosystems 7500 — Specialized for confirming the presence of a specific region (such as the transgene). Full-length assurance is needed separately

A basic test that measures 'how much is in it (concentration)' and 'how pure it is' by how light is absorbed. The amount of plasmid is obtained from absorbance at 260 nm.

Purity is seen by the A260/280 ratio. For pure double-stranded DNA, 260/280 is about 1.8 and 260/230 about 2.0 as a guide. Contamination with protein or organic matter shifts these values.

A step of a yardstick, so to speak, that provides the basis for deciding the dose. Because it can be measured easily, it is also routinely used as primary screening of purity.

Measure the amount (concentration) of plasmid and the A260/280 ratio, which indicates low protein contamination. For pure double-stranded DNA, 260/280 of about 1.8 and 260/230 of about 2.0 are the guide. It is the basis for dose design and also serves as the primary screening of purity.

Alternatives in practice

• Fluorescent dye method (dsDNA-specific) · Thermo Fisher Quant-iT PicoGreen dsDNA Assay Kit (P7589) + Qubit 4 / microplate reader — Quantifies dsDNA with high sensitivity without interference from RNA/ssDNA. Purity evaluation is separate
• Purity and content by AEX-HPLC/CGE · Agilent 1290 Infinity II Bio LC + DNAPac PA200, SCIEX PA 800 Plus — Evaluates purity and isoforms at the same time. Calibration and standards are required
• Absolute quantitation of specific sequences by qPCR · Thermo Fisher QuantStudio 5 — Absolutely quantifies the target copy number. A different indicator from total DNA amount

Plasmid is made inside E. coli, so a small amount of the bacterium's own genomic DNA can get mixed in. This is a safety concern, so it is strictly controlled.

The measurement is quantitative PCR (qPCR), which amplifies only sequences specific to the E. coli genome. Picking up even very small amounts with high sensitivity and pinning down the residual amount as a number is the practical mainstream.

In other words, a test that confirms 'how much DNA other than the target plasmid remains.' Only by showing it is within the specification can the lot proceed to release.

If genomic DNA from E. coli is mixed into the plasmid, it becomes a safety concern. This is quantified with high sensitivity to confirm that it is within the specification. Measuring the residual host genome by sequence-specific qPCR is the practical mainstream.

Alternatives in practice

• Digital PCR (ddPCR) · Bio-Rad QX200 Droplet Digital PCR System — Absolute quantitation without a standard curve and robust to inhibition. Throughput is somewhat low
• Estimation of host DNA in total dsDNA by fluorescent dye method · Thermo Fisher Quant-iT PicoGreen (P7589) — Simple, but not host-specific. Remains an indirect estimate
• Southern blot · Bio-Rad transfer/hybridization equipment — Also evaluates the fragment size distribution. Labor-intensive, and quantitative accuracy is inferior to qPCR

Residual protein derived from the host E. coli (HCP) is also subject to control as an impurity. It cannot be overlooked from the standpoint of immune reaction and quality.

For measurement, a sandwich ELISA using antibodies specific to host protein is used to quantify the total. It is a method that can capture many kinds of protein together.

An item confirmed at the lot release stage, showing it is within the specification. It serves as proof of how well the drug substance has been 'washed free of bacterial protein.'

Residual protein from E. coli is also controlled as an impurity. Total HCP is measured by ELISA using host-specific antibodies to show it is within the specification. An item confirmed at lot release from the standpoint of immunogenicity and quality.

Alternatives in practice

• Total protein quantitation (BCA/Bradford) · Thermo Fisher Pierce BCA Protein Assay Kit (23225) + plate reader — Simple, but not host-specific and with insufficient sensitivity at low concentrations
• HCP profiling by LC-MS/MS · Thermo Fisher Orbitrap Exploris / Q Exactive + Vanquish/EASY-nLC — Identifies and quantifies individual HCPs. Complements ELISA but the operating burden is heavy
• Indirect indicator of protein contamination by A260/280 ratio · Thermo Fisher NanoDrop One — Rapid but qualitative. Trace HCP cannot be detected

If E. coli RNA remains, it is mixed in when the amount of plasmid is measured, skewing the value and lowering purity. So the residual amount in the final product is confirmed.

In a process that does not degrade RNA with an enzyme, RNA is physically removed in the chromatography steps. How well that worked is cross-checked by capillary gel electrophoresis or agarose gel.

What is looked at is the presence and amount of the RNA band. By showing that none remains, the test assures both the correctness of quantitation and the purity of the product.

If RNA from E. coli remains, it skews plasmid quantitation and lowers purity. In an RNase-free process, RNA is removed by chromatography, so the residual RNA is confirmed in the final product. The presence and amount of RNA bands are evaluated by CGE or gel.

Alternatives in practice

• RNA peak separation by AEX-HPLC · Agilent 1290 Infinity II Bio LC + Thermo Fisher DNAPac PA200 — Separates and quantifies RNA and pDNA by HPLC. Easy to share equipment
• RNA-specific fluorescence quantitation (RiboGreen) · Thermo Fisher Quant-iT RiboGreen RNA Assay Kit (R11490) + plate reader — Selectively quantifies RNA. No size distribution information is obtained
• Indirect evaluation by A260/280 and A260/230 ratios · Thermo Fisher NanoDrop One — Rapid and simple but qualitative. Low-level residual RNA is difficult to detect

Endotoxin is a component from the outer membrane of E. coli and causes strong fever and immune reactions when it enters the body. It is controlled especially strictly for injectables.

The LAL method is used for measurement. Using the property that the horseshoe crab amebocyte extract reacts to endotoxin, very small amounts are quantified. Kinetic turbidimetric and chromogenic methods are used.

The specification is extremely low, and conformance is to levels such as generally below 0.1 EU/µg. Because it is directly tied to patient safety, it is a mandatory test that above all cannot be omitted.

Endotoxin from the outer membrane of E. coli causes strong fever and immune reactions, so it is strictly controlled. It is quantified by the LAL (horseshoe crab amebocyte lysate) method and brought into conformance with an extremely low specification according to use (generally below 0.1 EU/µg, etc.). A mandatory test directly tied to safety.

Alternatives in practice

• Recombinant factor C (rFC) method · Lonza PyroGene Recombinant Factor C Assay + fluorescence plate reader (Ex/Em 380/440 nm), or Fujifilm Wako PyroSmart NextGen — Sustainable, with no animal-derived material (independent of horseshoe crabs). Fluorescence detection
• Gel-clot LAL method · Charles River / Lonza gel-clot LAL reagents + heat block — Minimal equipment and suited to limit testing. Low quantitation and throughput
• Monocyte activation test (MAT) · Human whole blood/PBMC + cytokine ELISA (plate reader) — Also detects non-endotoxin pyrogens. Useful for overall pyrogenicity evaluation but cumbersome

A test that confirms that there are no living microorganisms in the final product (sterility) and how many microorganisms were present partway through the process (bioburden).

The method follows that specified in the pharmacopoeia. While grasping the microbial count during manufacturing, it confirms for every lot that no viable organisms are detected in the final product.

For an injectable nucleic acid therapeutic that goes directly into the body, sterility is directly tied to patient safety. That is why this is a step in which the prescribed procedures are strictly followed to assure quality.

Confirm that the final product is free of living microorganisms (sterility) and the microbial count during the process (bioburden). Because it is directly tied to patient safety as an injectable nucleic acid therapeutic, it is tested for every lot according to the pharmacopoeial methods.

Alternatives in practice

• Rapid microbiological methods (RMM) · bioMérieux BacT/ALERT 3D (automated growth detection) or Charles River Celsis (ATP bioluminescence) — Shortens judgment to a few days. Validation and demonstration of pharmacopoeial equivalence are required
• Bioburden by anaerobic/aerobic plate culture methods · Merck/Sartorius membrane filtration + agar plates — Standard for counting in-process bioburden. A different purpose from final sterility
• Process validation by filter integrity testing · Sartorius Sartocheck 5 Plus / Merck Integritest 5 — Assures 0.2 µm sterile filtration. Integrity is confirmed by bubble point/diffusive flow

A step that confirms the basic properties of appearance of the solution (whether it is clear, whether there is foreign matter or discoloration), pH and osmolality. It lays the groundwork as a drug product.

These govern stability and compatibility when put into the body (suitability for administration). Visual inspection and instrumental measurement are combined to confirm that quality is the same in every lot.

Unglamorous, but the foundation that holds down lot-to-lot variability. A confirmation item for the basic properties that support consistency as a drug product.

Check the appearance of the solution (clarity, foreign matter, discoloration), pH and osmolality. These are basic properties that govern stability and suitability for administration, and are confirmed for every lot by visual inspection and instrumental measurement. Items that assure consistency as a drug product.

Alternatives in practice

• Automated particle counting (light obscuration) · HACH/Beckman Coulter HIAC 9703+ liquid particle counter — Objectively quantifies insoluble particles (USP <788>). Complements visual inspection
• Vapor pressure osmometry · ELITechGroup/Wescor VAPRO vapor pressure osmometer — Measurable with a small sample. Different principle from freezing point depression
• Supplementary physical property confirmation by refractive index and conductivity · Mettler Toledo refractometer + conductivity meter — Simple monitor of concentration and salt concentration. For in-process confirmation rather than specification testing

Instead of plasmid, this step prepares a template containing the target sequence and primers that fix its two ends. E. coli is not used, so no extra bacterial-derived sequences are carried in.

If the ends of the primers are modified, it becomes easier to separate them from leftover template in later purification. The template may be either a synthesized gene fragment or a small amount of plasmid.

To make linear DNA by PCR without using plasmid (= E. coli), a template containing the target sequence (a synthesized gene fragment, or a small amount of plasmid) and a primer pair defining both ends of it are prepared. Some designs modify the ends of the primers with phosphorothioate and the like to give exonuclease resistance and make the later selective purification easier. The sequence and purity of the template and the primer quality determine the fidelity of amplification and the final purity.

Alternatives in practice

• Using synthesized long DNA (gene fragment) directly as template · Gene synthesis such as Twist Bioscience/GenScript — Prepares the template without going through plasmid construction. Feasibility depends on sequence length and GC content

A step that PCR-amplifies the prepared template and primers with a polymerase that has a proofreading function, rapidly increasing linear DNA of the target sequence. Without waiting for cell culture, large amounts are obtained in hours to days.

Because bacteria are not used, it is fast and no extra sequences get in. On the other hand, fidelity that suppresses sequence errors and how evenly quality is maintained when the quantity is increased are where skill shows.

The template is PCR-amplified with a high-fidelity DNA polymerase having proofreading activity, giving a large amount of linear double-stranded DNA of the target sequence. Because no cells or fermentation are used, the advantage is speed, with large quantities obtained in hours to days, and it is used for IVT templates of mRNA, DNA medicines and vector starting materials. Fidelity (error rate) and, at scale, control of yield, uniformity and template carryover are the issues.

Alternatives in practice

• Cell-free DNA specialized for IVT templates (LineaDNA, etc.) · Applied DNA Sciences/LineaRx — PCR-based production under contract in GMP. Procures linear DNA template without owning equipment

In the enzymatic synthesis route, a small circular template containing the target sequence and a 'marker for closing the ends (recognition sequence)' is prepared. It is denatured to be ready for the next isothermal amplification.

Only a tiny amount of template is enough. From here on, no E. coli is used, and the lightness of this method is that amplification proceeds all at once with enzymatic reactions alone.

In the enzymatic synthesis (doggybone, dbDNA) route, a small circular template containing the target sequence and the recognition sequence of protelomerase (about 56 base pairs) is prepared and chemically denatured so that it can be subjected to rolling circle amplification (RCA). Only a very small amount of circular template is needed, and a feature of this route is that amplification to large quantities then proceeds by cell-free enzymatic reactions alone.

The polymerase phi29 is very strong at advancing while unwinding double strands (strand displacement), and keeps reading around the circular template to make a long DNA in which the target sequence is strung like beads. This is rolling circle amplification (RCA).

It proceeds just by holding the temperature constant, with no need for E. coli culture or plasmid extraction. It also has a proofreading function, so it can earn volume while keeping the sequence accurate.

Using the strong strand displacement activity of phi29 DNA polymerase, the circular template is amplified isothermally (RCA) to make a long linear double-stranded DNA (concatemer) in which the target sequence is repeatedly joined. It scales easily as an isothermal, cell-free process, and only a trace of template is needed. Because phi29 has 3'→5' proofreading activity, it can amplify in large quantities while maintaining fidelity.

Alternatives in practice

• primer-free enzymatic amplification (TruePrime) · 4basebio (hpDNA/opDNA/osDNA) — Synthetic DNA manufactured by primer-free enzymatic amplification. Contract/platform

Protelomerase is an enzyme that cuts a defined sequence within long strung-together DNA and folds back the cut ends and closes them by covalent bonds. A linear DNA with both ends sealed, in a distinctive 'bone (doggybone)' shape, is formed.

With closed ends, even if leftover 'junk' DNA (with open ends) is chewed away later with an enzyme, only the target remains undamaged. Another strength is that it can enclose only the target sequence without including extra sequences.

Protelomerase (TelN, etc.) cleaves the recognition sequence in the concatemer and turns the cut ends into hairpin ends closed by covalent bonds. This shapes it into linear DNA with both ends closed (doggybone/dbDNA). Closed ends are resistant to exonuclease, so in later purification they can be selectively separated from remaining open circular and linear DNA. The advantage is that only the target sequence can be enclosed in a minimal construct.

Alternatives in practice

• Closure on a contract platform (dbDNA, etc.) · Touchlight dbDNA/Aldevron Alchemy — Contract manufacturing of cell-free DNA including closure. An option that avoids handling enzymes in-house

Exonuclease is an enzyme that trims DNA from its ends one nucleotide at a time. The target DNA with closed ends is not trimmed, and only the leftovers with open ends (fragments of template and backbone) are degraded.

In the PCR route, too, leftover primers and single-stranded DNA are cleaned up at this stage. The convenience of the end-dependent property is that it can protect the target and erase impurities selectively.

While leaving the target linear DNA, unneeded residual DNA is selectively degraded by enzyme. In the enzymatic synthesis route, closed-end doggybone DNA is resistant to exonuclease, while remaining open circular and linear template and backbone-derived DNA are degraded from the ends, so only the target can be selected and left. In the PCR route, too, it is used to remove residual primers, template and single strands.

Alternatives in practice

• Combined use of an internal-cleaving nuclease (Benzonase) · Merck Benzonase, etc. — Broadly reduces residual nucleic acids to small fragments. It has no end-dependent selectivity, so its use is distinguished

A step that removes leftover enzymes, nucleotides, fragments and endotoxin and finishes at drug substance quality. DNA is captured by anion exchange or monoliths, using its negative charge, and ultrafiltration adjusts the concentration and the contents (buffer).

Because E. coli is not used, there is less host-derived dirt, but it is confirmed that the enzymes and template used and endotoxin have been properly removed. Finally it is sterile filtered and becomes a template for IVT, a starting material for vectors, or the drug substance of a DNA medicine.

Enzymes, nucleotides, degradation fragments and endotoxin are removed to finish at drug substance quality. Anion exchange (AEX) using the negative charge of DNA and monoliths capture and polish, and ultrafiltration/diafiltration (UF/DF) concentrates and exchanges the buffer. Because no cells or fermentation are involved, host-derived impurities are few, but confirmation of removal of residual enzymes (HCP equivalent), template and endotoxin is needed. Finally it is sterile filtered to make the drug substance for IVT templates, vector starting materials and DNA medicines.

Alternatives in practice

• Contract manufacturing of cell-free DNA (including purification) · Touchlight/Applied DNA, LineaRx/Aldevron/GenScript — Contract service covering amplification through closure, purification and specification testing. Supplied in GMP grade