Inquiry Basket
High-performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis (PAGE) are two common techniques used to purify oligonucleotides. These purification systems efficiently separate full length product from failure sequences and side products resulting from chemical synthesis. This separation is important because the synthesis of oligonucleotides is not 100% efficient and produces truncated failure sequences. Regardless of the purification strategy, high purity products can be attained for many applications.
Purification of oligonucleotides is essential, given that the crude synthesis yields a mix of undesirable byproducts, including incomplete sequences and deprotected nucleotides. Byproducts include deletion mutants and modified nucleotides that interfere with migration during purification. If not purified these byproducts can interfere with hybridization, decrease enzyme activity, and lead to erroneous quantitative interpretation. Purification strategies also affect the success of subsequent applications for research use, therapeutics, or diagnostics.
Fig. 1 Overview of the TOs processing with upstream and downstream operations.1,5
Damaged oligonucleotides in raw samples can cause problems in a variety of uses. Oligonucleotides that are incomplete can still find and bind to their target sequence. As they compete for hybridization with probes designed for that target sequence, the signal will be weaker than expected. This can lead to false negatives in many applications. In PCR applications incomplete products can act as additional primers leading to spurious amplification and false positives. Degradation can also affect quantitative applications like qPCR where competition can lead to skewed standard curves and Ct values resulting in miscalculations of how many copies of a target are present. If modified oligonucleotides are used, degradation products may have different spectroscopic signatures and/or different sensitivities to enzymatic hydrolysis which can interfere with the expected kinetics. If the oligonucleotide is being used as a drug, degradation products can cause undesirable side effects. Some degradation products have been shown to trigger the immune response by activating toll-like receptors, leading to inflammation.
Oligonucleotide purification techniques can be quite simple (e.g. desalting) or very sophisticated (chromatography and/or electrophoresis with high resolving power). Regardless of the complexity of the purification strategy, all synthesis reactions are stepwise processes that do not go to completion. Therefore the reaction mixture must be purified to remove residual coupling reagents and salts, but also to remove failure sequences that result from incomplete synthesis chemistry. The simplest purification separates all species based on size only and will leave intact failure sequences that are shorter than the desired product (e.g. size-exclusion chromatography or precipitation). More sophisticated purification procedures separate material based on hydrophobicity, charge, or size. These procedures can be used to specifically purify full length (desired) sequence from deletion products. The level of purification required for an oligonucleotide depends on how the oligonucleotide will be used. Many routine amplification reactions do not require highly purified oligonucleotides, however therapeutic, diagnostic and structural applications typically require highly purified material with almost all failure sequences removed.
Fig. 2 Process overview of oligonucleotide manufacturing operations.2,5
Desalting is the simplest level of purification and relies upon molecular sieving to remove small molecules such as protecting groups, reagents used in the synthesis and salts, but does not separate fragments from full length material. Allowable products include truncated molecules as well as deletion mutants. As such, desalting is often only sufficient for preparing templates for routine PCR amplification or sequencing reactions where inclusion of these impurities will not negatively impact performance. Reversed-phase chromatography, ion-exchange chromatography, or denaturing PAGE purification provides a much higher level of purity by exploiting the physicochemical properties of the desired full-length product versus truncated failure sequences. These purification schemes effectively separate truncated fragments and chemically modified side-products from the target material creating homogenous preparations ideal for use in quantitative experiments, structural characterization or for therapeutic use where the activity of the product is sequence dependent.
| Purification Method | Separation Principle | Impurities Removed | Suitable Applications |
| Desalting | Size exclusion | Salts, small molecules, reagents | Routine PCR, sequencing, preliminary screening |
| Cartridge purification | Reverse-phase hydrophobicity | Failure sequences, salts | Cloning, standard PCR, labeling reactions |
| HPLC | Hydrophobicity or ion-exchange | Failure sequences, deletions, modified byproducts | Quantitative assays, therapeutics, diagnostics, structural studies |
| PAGE | Size and conformation (denaturing) | All truncates and deletions | Crystallography, gene synthesis, high-fidelity applications |
Table 1 Comparison of Oligonucleotide Purification Methodologies
The most commonly used technique for purifying synthetic oligonucleotides is HPLC. The basis of purification by HPLC involves separating molecules based on their affinity to column matrices. As such, HPLC allows separation of full-length oligonucleotide products from failure sequences. Typically, HPLC purification consists of reversed-phase and ion-exchange modes to separate incomplete extension fragments, deletions, and chemically modified products.
Ion-pair reversed-phase chromatography with RP-HPLC is perhaps the most widely practiced form of oligonucleotide purification. Because of their phosphate backbone, oligonucleotides are negatively charged. Thus, they are repelled by the hydrophobic surface. Adding a bit of an ion-pairing reagent like triethylamine or hexafluoro-2-propanol, which has those alkylammonium groups, helps even out the oligonucleotide's charge for a short while. This causes the oligonucleotide to stick to the column. Next, a gradually increasing amount of organic solvent is used to remove the sample. Earlier-eluting compounds include shorter sequences, more hydrophobic variants, and those with chemical modifications, while the longer and fully formed product comes later. Columns can be heated and run under denaturing conditions to prevent secondary structure from forming on the oligonucleotide which would negatively effect resolution. These columns can typically separate sequences that differ by a single nucleotide when baseline resolution is achieved. Ion pair HPLC can also be used to purify oligonucleotides with a variety of chemical modifications such as fluorescent tags, backbone modifications, and attached ligands.
Oligonucleotides are polyanionic compounds. They can therefore be separated by anion exchange chromatography based on differences in charge density between intact product and shorter failure sequences. Longer oligonucleotides will have a stronger affinity for the anion exchange material (dependent on the number of phosphoryl groups along the chain). Elution from anion exchange columns is easily accomplished by increasing salt concentrations. Longer chains will tend to elute at higher salt concentrations than shorter species. Since phosphorothioate oligonucleotides have both charge heterogeneity as well as altered hydrophobic characteristics as compared to unmodified oligonucleotides, anion exchange chromatography can offer a convenient orthogonal separation method to reversed-phase chromatography. Rapid, inline flow-mode anion-exchange chromatography using membrane technology allows for fast flow rates (free of diffusion problems associated with particle-based chromatography) which is ideal for large scale manufacturing applications. By far, the greatest advantage of anion exchange over reversed-phase is the ability to easily remove shorter failure sequences and many process-related impurities with excellent recovery. Resolution can be poor for longer chains as well as chromatographic separation of modified sequences when compared to reversed-phase chromatography.
Benefits of HPLC include high resolution (down to single nucleotide differences), automation (little human interaction), easy scale-up from analytical to process scale (methods often translate easily between the two scales), and combination of desalting, detritylation, and purification all in one step which can simplify the overall process and increase yields. Drawbacks to HPLC include: resolution dependent on length (longer products generally result in broader peaks with lower resolution), secondary structure stability (observed with sequences such as GC-rich and self-complementary sequences) which can lead to abnormal retention times, and instability of some fluorophores or adducts to conditions used in the mobile phase or at the higher temperatures needed to denature secondary structures. Gradient conditions, types of ion-pairing reagent used, and flow rates all need to be optimized which can be laborious.
Polyacrylamide gel electrophoresis (PAGE) is a basic method of purification and analysis of synthetic oligonucleotides. It separates full length product from synthetic side products on the basis of size. This is typically done in denaturing gel conditions. Because all nucleic acid molecules possess the same charge per mass ratio, the speed at which they move through a PAGE gel is a function of size. PAGE can resolve fragments that differ by a single nucleotide. PAGE is frequently used for purification of longer oligonucleotides or when the highest degree of purity is required. Almost any chemistry can be PAGE purified including modified bases and backbones. However recovery and scalability are typically inferior to chromatography based purification methods.
PAGE purification exploits electrophoretic mobility of negatively charged oligonucleotides through a polyacrylamide gel under denaturing conditions. Urea or formamide denatures secondary structure so that electrophoretic mobility depends only on molecular weight. Desired full-length product will move more slowly than shorter failure sequences allowing them to be physically separated into bands. The desired band is cut out of the gel and oligonucleotide is removed by passive diffusion or electroelution into an aqueous buffer followed by desalting and concentration.
Strengths of PAGE are higher resolution (often single-basepair resolution), ability to be used on fragments of all sizes from small PCR primers to megabase oligonucleotides required for chemical gene synthesis and PAGE can be used with chemically modified oligonucleotides. PAGE purification can be routinely performed to yield DNA fragment purities greater than 95% which is often required for challenging uses of oligonucleotides such as crystallography or drug development. Weaknesses of PAGE include requirement for hands-on time to manually cast gels and run electrophoresis, manually slice out bands then extract purified DNA from gel. These steps can take days to complete. DNA fragment recovery tends to be low (often less than 50%) due to gel extraction limited by diffusion as well as losses incurred during each manual handling step. PAGE is not easily scalable for manufacturing uses. It is not recommended for some fluorophores or modifications that are incompatible with acrylamide or other chemicals used in gel preparation.
HPLC and PAGE use completely different technologies for purification of oligonucleotides. They separate species based on different characteristics, have very different workflows, and their use can be dependent on the intended application. Both can be used to purify oligonucleotides to very high purities, but differ in terms of resolution, scale-ability, throughput and cost.
| Performance Parameter | High-Performance Liquid Chromatography | Polyacrylamide Gel Electrophoresis |
| Resolution capability | High; single-base for short sequences | Very high; single-base for all lengths |
| Purity achieved | High to very high | Very high (premium grade) |
| Scalability | Excellent (analytical to manufacturing) | Limited (laboratory scale) |
| Throughput | High (automated processing) | Low (manual-intensive) |
| Turnaround time | Rapid (hours) | Extended (days) |
| Recovery yield | Moderate to high | Lower (diffusion-limited extraction) |
| Length compatibility | Optimal for short to medium | Superior for ultra-long sequences |
Table 2 Comparative Analysis of HPLC and PAGE Purification Methods
Both have capabilities of providing very high purities suitable for most purposes. PAGE achieves this by separating molecules according to size using denaturing conditions so that secondary structure does not affect the mobility. PAGE has a resolution of single base pair substitutions throughout the entire length range allowing separation of full-length product from deletion/loading failures that are one base shorter. Ion pair reversed phase HPLC also separates oligonucleotides cleanly. However the resolution becomes poorer with increasing chain length. Therefore HPLC purification still degrades shorter oligonucleotides, as well as modified oligonucleotides, yet still results in a very clean final product usable for most purposes, though additional cleanup may be necessary to attain highest purities of very long oligonucleotides.
A key difference between these techniques is scalability. Liquid chromatography methods developed on analytical scales can be directly scaled up for use in preparative or manufacturing processes without significant re-development. HPLC systems can be automated to analyze many samples in succession with little intervention, making them ideal for high-throughput or commercial manufacturing processes. Polyacrylamide gel electrophoresis based purification is limited in scale to lab environments because making, running, visualizing, cutting, and extracting bands from polyacrylamide gels is difficult or impossible to automate. Gel based methods also require significant hands-on time for each purification, limiting throughput. Chromatography can be scaled by using columns with larger diameters and can process larger sample volumes at increased flow rates.
In terms of costs, time and labor, there are pros and cons for each method. When using HPLC, although there is a large up-front cost for equipment, the purification can be completed within a few hours. Since HPLC is automated, if purifying large numbers of samples, the cost per sample can be decreased. However, there are operating costs that must be done with each run. Solvents can get expensive and the column may need to be replaced occasionally. Purification by PAGE is laborious and can take up to a couple days to finish. Since there is more manual work involved, the cost for labor can add up. There is not a large initial cost like with HPLC, because all you need is an electrophoresis chamber. First you have to allow time for the gel to polymerize. Then you need to allow time for the sample to run on the gel, visualize the proteins, cut out the protein and elute out your protein overnight by diffusion or electroelution. Therefore, if you have a time sensitive process, HPLC might be your best bet. However, if you just need to purify small amounts for standard lab use, PAGE might be cheap. On an industrial scale, HPLC can be easily scaled up.
The choice of purification technique is dependent on the intended use of the oligonucleotide product. Methods with higher resolution may be preferred when oligonucleotide quality is critical; however, these methods may also have tradeoffs in time, throughput capacity, and recovery. For research use only applications where the oligonucleotide is being used for regular PCR or sequencing analysis, truncation can generally be accepted which allows for quicker desalting or purification cartridges which only remove low molecular weight impurities and do not clean up failed sequences. In diagnostic use, it is important to achieve cleaner oligonucleotides that will allow for consistent quantitative performance as well as eliminating false positives created by truncated products. Clinical use oligonucleotides typically require higher resolution techniques such as chromatography or electrophoresis with stringent documentation and batch-to-batch consistency. In addition, these methods typically provide a higher purity product. The choice of the purification method may also depend on the chemistry of the oligonucleotide being purified. Some modes of separation may be incompatible with certain chemistries or certain lengths and sequences.
Dependent upon the intended use of the oligonucleotide, different purity criteria are required and thus different purification methods are employed. Laboratory or research-grade oligos which will be used in reactions frequently performed in molecular biology labs such as PCR, cloning or hybridization probing can be purified by desalting or cartridge purification. Both methods remove most synthesis reagents and low molecular weight impurities but do not remove truncated sequences which are typically inconsequential to most applications. Oligos intended for diagnostic use such as qPCR or clinical genotyping assays require greater purity to ensure consistency from experiment to experiment, accurate quantitation and freedom from contaminants that could produce spurious signals. Such purity is achieved using reversed-phase or ion exchange high-performance liquid chromatography (HPLC) which separates full length oligo from truncated species. Clinical or therapeutic oligonucleotides must be purified on preparative scale under quality control procedures with comprehensive documentation, validated methods of analysis, and identified impurities. As consequences of failure grow more severe, the stringency of purity and documentation increases accordingly. Therapeutic uses would necessitate significant investments in equipment and quality control that would not be cost-effective for general laboratory use.
The structure of your oligo will also determine how you purify your product. This can include the backbone (bridged vs. unmodified phosphates), any conjugates at the 3' or 5' end, or overall length. If you have a standard DNA sequence that does not have more than a few modifications, reversed-phase chromatography or denaturing gels can easily purify your oligo and the decision of which purification strategy to use can be based on the length of the oligo or the purity needed. If you sequence is heavily modified with things like phosphorothioates, fluorophores, or other hydrophobic conjugates the behavior of your oligo during chromatography can change and you may need specialty ion-pairing reagents or mobile phase additives. If your oligo product is very hydrophobic you may experience adsorption to the stationary phase or product aggregation. Long oligos (generally >100nt) can be difficult to separate cleanly by chromatography because of increasing secondary structure potential and less difference in size between full-length and n-minus-one products. This usually means you'll need to use gel purification which has a lower recovery but can better resolve these products.
The method chosen for purification determines what quality control testing can be documented for oligonucleotides intended for research, diagnostic or therapeutic use. Any purification method results in characteristic impurities and carryovers which can be assessed using orthogonal methods. Chromatography systems, like high-performance liquid chromatography, can offer purity measurements as well as fractions collection to assist in further characterization. Gel purification methods typically offer higher resolution purification. Validating and documenting analytical testing from gel purification can be challenging. Documentation of the purification method including column or gel specifications, gradient or elution conditions and selected fractions should be included in oligonucleotide batches that are near translational or clinical use.
Chromatography easily couples with detectors (UV, MS) and fraction collectors, allowing further characterization. Also, HPLC methods such as RP-HPLC and IEC provide quantitation and the ability to set hard cut-off values for purity that allow for unbiased decisions to release or hold a batch based on acceptance criteria. Additionally, the fully instrumented approach of chromatography allows for automatic recording of the retention times, UV trace, and fraction cutoff times needed for documentation purposes. Gel electrophoresis typically involves visual inspection of the gel followed by manual cutting and extraction of the desired protein. This provides documentation challenges; however, capturing an image of the gel can verify purity if validated properly. Either way, one should consider how the protein will be analyzed during method development. If the protein will be used for therapeutic purposes, the method of purification should allow for validation of the methods used to assess protein identity, purity, and potency.
Scale-up of purification processes is another consideration that affects Quality Control Strategy as well as regulatory filings when taking an application from Research & Development into commercial production. Chromatography lends itself easily to scale-up (moving from analytical to prep size) with no change in separation principles or quality attributes, allowing for ease of process validation by proven equivalence. New technologies allowing continuous multi-column chromatography, such as multicolumn countercurrent solvent gradient purification, can be performed at the process scale allowing uninterrupted processing with automated recycling of overlapping fractions resulting in highest yield/purity with no compromise and robust data for regulatory filing purposes. Techniques utilizing gels can be difficult to scale up and manual gel extraction techniques would not be easily translated into a manufacturing process. Gel extraction is also more susceptible to operator bias. Validation of scaled-up purification requires extensive documentation including standard operating procedures, in-process controls, acceptance criteria and successful demonstration of consistency over multiple production runs. Chromatography lends itself to process analytical technology (PAT) platforms allowing for online monitoring.
| Purification Method | Analytical Output | Documentation Capability |
| Reversed-phase HPLC | Quantitative purity profile, peak integration | Electronic records, automated data capture |
| Ion-exchange HPLC | Charge-based purity assessment | Comprehensive elution documentation |
| Denaturing PAGE | Visual purity assessment | Photographic documentation, manual records |
| Cartridge purification | Qualitative purity estimate | Basic batch records |
Table 3 Quality Control Implications of Purification Method Selection
Selecting the appropriate purification method is critical to achieving the required balance between purity, yield, scalability, and cost. The choice between HPLC and PAGE depends on oligonucleotide length, sequence complexity, modification density, and application sensitivity. Our oligonucleotide purification capabilities include:
By integrating tailored purification strategies with analytical confirmation, oligonucleotides can be aligned more precisely with performance requirements across research and regulated applications.
In many cases, assay sensitivity, reproducibility issues, or inconsistent experimental results can be linked to insufficient purification or inappropriate method selection. You may benefit from reviewing your purification approach if your project involves:
An informed purification strategy can improve assay performance while maintaining efficiency and scalability. Contact our team to discuss your oligonucleotide purification requirements and identify the most appropriate HPLC or PAGE strategy for your specific application.
References
