Quality control

Reversed-phase chromatography is an efficient method for the purification and analysis of synthetic oligonucleotides. Reversed-phase chromatography uses a hydrophobic stationary phase. Oligonucleotides and impurities that arise during their synthesis are then eluted at different times due to their differences in hydrophobicity. This is achieved by a buffered solvent or mixture of solvents called the mobile phase.

At Ella Biotech we use RP-HPLC for the purification of oligonucleotides as well as for quality control.

Ion chromatography (or ion-exchange chromatography, short: IEX) separates oligonucleotides on the basis of negative charge differences based on the number of phosphodiester groups at the backbone of an oligonucleotide. An efficient separation of longer, more charged oligonucleotides is accomplished on a cationic stationary phase due to an increase of the eluting strength of the mobile phase. This method is an effective technique and orthogonal to the method of reversed phase chromatography.

At Ella Biotech we also use IEX-HPLC for the purification of the oligonucleotide as well as for quality control.

Matrix-assisted laser desorption/ionization (MALDI) is a mild ionization technique used in mass spectrometry, allowing the analysis of oligonucleotides.

The MALDI-TOF (TOF=time of flight) is suitable to resolve oligonucleotides up to 50-60 bases in length (up to about 18.000 Daltons). It is a fast analysis technique suitable for efficient identity control of oligonucleotides that allows us to provide large quantities of oligonucleotides within a few working days.

At Ella Biotech, MALDI-MS is the method of choice for our internal high throughput quality control. We check oligonucleotides of each production run as well as all produced modified oligos and dual-labelled probes.

Electrospray ionization (ESI) mass spectrometry uses a small amount of the synthetic oligonucleotide that is ionized to multiple negatively charged ions/species, which are propelled into a mass detector/analyzer. The ESI-MS technique has the advantage that the oligonucleotide usually does not fragment during the process of ionization and is suitable to analyze longer oligonucleotides (up to ~150 bases in length).

The raw data of the multiple charged species are then processed using a deconvolution algorithm that takes all of the peaks present in the trace. The obtained mass can be compared to the known mass (theoretic mass) of the oligonucleotide sequence.

At Ella Biotech, ESI-MS is used especially for oligonucleotides longer than 30mers and for all oligonucleotides produced according to ISO 13485:2016.

Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate oligonucleotides according to their electrophoretic mobility. The mobility of an oligonucleotide depends on its length, conformation and charge.

At Ella Biotech, we use PAGE mainly for quality control as well as for purification on customers´ request.

We recommend PAGE analysis and purification for long oligos, where other purification methods may present difficulties. PAGE purification typically results in oligonucleotides of very high purity, however this comes at the expense of yield.

The final analysis of dye labelled oligonucleotides with PAGE is done on a fluorescent scanner to see possible side products.

Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels.

At Ella Biotech, we use CE for the quality control of modified oligonucleotides, mainly on customer request.