Polynucleotides — long-chain DNA fragments, commonly of salmon origin and supplied as sodium polynucleotide or PDRN — have become a leading biotechnology-derived active in aesthetic products, above all injectable skin boosters and mesotherapy solutions. Their quality rests on three linked attributes: the polynucleotide content, the molecular weight distribution of the polymer chains, and the microbiological and impurity burden carried over from biotechnological manufacture. This article arranges testing practice for such products into six modules: analytical principle and mechanism; sample collection and pretreatment; content assay by HPLC and UV spectrophotometry; molecular weight analysis by gel electrophoresis and SEC-HPLC; co-tested parameters covering endotoxin, sterility and residual proteins; and assay performance with typical application scenarios. Taken together, these assays generate the data used for raw-material qualification, batch release and stability evaluation, so that conformity of polynucleotide-based aesthetic products can be judged against product specifications and pharmacopoeial expectations.
Principle & mechanism
Polynucleotide actives in aesthetic products are linear DNA polymers composed of deoxyribonucleotide units, commonly presented as sodium salts in sterile injectable vehicles. In skin tissue, the proposed mechanism involves nucleotide salvage and activation of adenosine A2A receptors, an effect associated with fibroblast proliferation and extracellular matrix remodelling. The claimed activity therefore depends on chain length and purity rather than on a single defined molecule, and this molecular character defines the analytical strategy. Conjugated bases in the polymer absorb ultraviolet light near 260 nm, which permits spectrophotometric quantification. The uniformly anionic phosphate backbone interacts with strong anion-exchange stationary phases, allowing chromatographic separation of the polymer from oligonucleotide and mononucleotide species. Chain dimensions, in turn, control mobility in agarose gels and elution volume on size-exclusion columns. Testing consequently combines identity and content measurement with structural characterization, and mechanism-based specifications concentrate on molecular weight range, polynucleotide fraction and absence of degradants.
Sample types, collection and pretreatment
Samples submitted for analysis fall into three categories: bulk drug substance (sodium polynucleotide or PDRN powder), intermediate concentrates, and finished products such as pre-filled injectable solutions or gels. Bulk powders are weighed and dissolved in nuclease-free water or neutral buffer to a defined concentration, with gentle mixing at low temperature where dissolution is slow. Injectable finished products are drawn from intact containers under aseptic technique, and aliquots for microbiological testing are taken before any chemical manipulation. Because nucleases degrade the polymer, all reagents are nuclease-free, EDTA may be added where the downstream method tolerates it, and samples are held at 2–8 °C and processed promptly. High-molecular-weight grades can show elevated viscosity; dilution or brief mild warming yields homogeneous solutions. Particulates are removed by centrifugation or 0.45 µm filtration, and membranes are checked beforehand for absence of polynucleotide adsorption. Every pretreatment step is documented so results trace back to a defined preparation scheme.
Polynucleotide content — HPLC and UV spectrophotometry
Content testing answers two questions: how much polynucleotide the product contains, and how much of that material remains in polymeric form. UV spectrophotometry serves as the screening method. The sample is diluted until the absorbance at 260 nm falls inside the linear range, and concentration is calculated with established conversion factors for nucleic acid salts. The A260/A280 ratio, close to 1.8 for clean DNA preparations, flags protein contamination, while an elevated A230 reading points to phenolate or guanidine carry-over. HPLC supplies the orthogonal confirmation. Strong anion-exchange columns eluted with a sodium chloride gradient retain the polymer according to charge density and resolve it from short oligomers and free nucleotides; detection at 260 nm against an external polynucleotide standard gives the assay result. Reversed-phase methods are reserved for monitoring monomeric nucleotide impurities. During validation, agreement between the two techniques within the set tolerance demonstrates that the spectrophotometric value is not inflated by low-molecular-weight absorbing species.
Molecular weight distribution — gel electrophoresis and SEC-HPLC
Molecular weight distribution governs the rheological behaviour of the injected product and its residence time in tissue, so laboratories treat it as a critical quality attribute. Agarose gel electrophoresis offers the visual screen: fragments migrate according to size against a DNA ladder spanning the declared range, and smeared or diffuse bands expose degradation or broad polydispersity. Capillary gel electrophoresis shortens run times and improves resolution at the low end of the range. The quantitative profile comes from size-exclusion HPLC. Chains separate by hydrodynamic volume on the column, UV or light-scattering detection records the elution profile, and calibration with nucleic acid standards of assigned size yields the average molecular weight and the polydispersity index. The column is chosen so the declared range lies inside the calibration curve, and injection viscosity is kept low to prevent artificial peak broadening. A drift in the distribution triggers review of storage conditions and expiry dating.
Co-test parameters — endotoxin, sterility, residual proteins
Because polynucleotide skin boosters are injected into the dermis, microbiological and impurity controls carry the same weight as the potency assay. Bacterial endotoxin is measured by limulus amebocyte lysate procedures — gel-clot, kinetic turbidimetric or chromogenic — once product-specific validation has excluded inhibition or enhancement of the reaction. Results are compared with the limit derived from the maximum injected dose. Sterility testing follows the membrane filtration technique whenever the product is filterable, with soybean-casein digest and fluid thioglycollate media incubated for the pharmacopoeial observation period. Residual proteins from source tissue and extraction steps are quantified by Bradford or BCA colorimetric assays, with an interference check, since polynucleotide salts can alter dye responses. Routine additions cover pH, osmolality and residual solvents; salmon-derived material may call for residual-antibiotic screening where the process history indicates it. Third-party laboratories report these results alongside content and molecular weight data in one release package.
Assay performance and application scenarios
Method performance is documented before routine use. Key metrics include specificity against formulation excipients, linearity across the working concentration range, precision expressed as relative standard deviation of replicate preparations, accuracy checked by standard addition, and detection and quantification limits for impurity monitoring. For chromatographic and electrophoretic runs, system suitability criteria — resolution of adjacent peaks or bands, retention-time consistency, standard-curve fit — are fixed in the validation protocol and verified in each analytical sequence. Acceptance criteria follow the product specification and, where applicable, the pharmacopoeial chapters for parenteral preparations. Typical applications include incoming inspection of bulk polynucleotide, in-process control during formulation, batch release of finished injectables, shelf-life and accelerated stability studies, and comparison of suppliers or reformulated grades. When a deviation appears — a falling content, a shifting molecular weight profile, an out-of-trend endotoxin value — the same analytical package locates the affected stage. The investigation may span raw-material hold time to terminal sterilization, and repeat testing shows whether corrective action has restored control.