Laboratory Testing Blog

Chromatographic Method Analysis for Oligonucleotides Using UPLC and UPLC-MS/MS Detection

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Typical oligonucleotides used as therapeutics or potential therapeutic compounds are 15 to 30 nucleotides (nt) long.  Oligonucleotides <15 nt can be readily resolved by HPLC technology.  However, the separation of longer sequences are more challenging.  Ion-pair reversed-phase (IP-RP) HPLC has been traditionally used for oligonucleotides analysis.  The traditional IP-RPLC eluent system typically uses TEA or TEAA or HFIP and the ion-pairing agent triethylammonium ion and a C18, column at 60^oC.  Under such conditions, the HPLC column's hydrolytic stability becomes crucial and is important to prevent the potential contribution of the oligonucleotide secondary structure from impacting retention.  To overcome this, Waters has an ACQUITY UPLC BEH column that has demonstrated excellent performance for oligonucleotides of similar structures.

Column separation performance in gradient elution mode is normally measured as peak capacity.  The peak capacity represents the maximum theoretical number of peaks that can be resolved within the gradient time.  For oligonucleotides in a traditional HPLC method, the target compound (N), in many cases, elutes close to shorter species (N-1, N-2, etc.) and the separation selectivity cannot be easily altered.  Therefore, column peak capacity is critical.

From previous experience, the ACQUITY UPLC OST columns have high peak capacity (1.0 mm or 2.1x 50 mm packed with 1.7μm C18 smaller sorbent) and have demonstrated improvements in resolution and faster analysis.  Routinely, 15 to 25 nt can be baseline-resolved within 10 minutes compared to 60 minutes in traditional HPLC analyses.

Undertaking method development work for an oligonucleotide separation usually includes an optimization of gradient slope and initial mobile phase elution strength.  Phosphorothioate oligonucleotides are more difficult to analyze than phosphorodiester ones.  When replacing an oxygen atom in the oligonucleotide backbone for sulfur, multiple diastereomers can be created.

TEAA, an ion-paring reagent, is useful for phosphordiester oligonucleotides.  However, it fails when it is applied for separation of phosphorothioate oligonucleotides.  Recently, a novel ion-paring buffer was introduced for efficient analysis of therapeutic oligonucleotides.  The buffer is comprised of triethylamine TEA, an ion-paring agent and hexafluoroisopropanol (HFIP) a volatile weak acid (pH ~8).  This ion-paring system is compatible with both UV and MS detection systems.  TEA-HFIP ion-pairing buffer yields more consistent and predictable oligonucleotide retention behavior as compared to TEAA.

Method optimization parameters should include:

• Identifying a suitable initial gradient strength, starting with a scouting gradient

• Adjusting the gradient slope to achieve a desirable separation; in general, a shallower gradient provides increased resolution

• Adjusting the flow rate to achieve faster analysis while maintaining gradient column volumes

Since the gradient slope stays unchanged, the high resolution separation should be preserved with the possible exception of the early eluting peaks.

When using UPLC detection, in order to obtain additional confirmation for identity of parent and fragments/impurities (N ±X), a UPLC-UV method should be converted to a UPLC/MS/MS method.  However, if significant structural information of targeted oligonucleotides it is essential, then using Q-TOF MS technology with high mass accuracy will be required.

There are a very limited number of existing analytical technologies or methodologies available that can be simply employed to achieve the technological objectives of this kind of project.  The technologies such as HPLC, LC/MS/MS or EC that traditionally have been employed for medium to large size protein characterization may not yield desirable results.  Therefore, if the oligonucleotide is complex. attempts to achieve ether acceptable separations of the related substances or to quantify the active molecule with desired sensitivity can fail.

Review of existing published scientific literature describing synthetic protein molecules will suggest several available analytical methodologies including HPLC, UPLC, sodium dodecyl sulphate, Polyacrylamide Gel Electrophoresis (SDS PAGE) and Enzyme-Linked ImmunoSorbent Assay (ELISA).  If the goal is to select and establish an appropriate analytical method for a drug product that suppresses the expression of mutated cancer cells in cancer patients, then UPLC technology, in conjunction with specific ion per interaction mechanism should be evaluated with optimal chromatographic conditions selected for method development.  By pursuing this approach, a new Ultra Performance Liquid Chromatographic (UPLC) method can be developed and validated to successfully separate and quantify the active ingredient and all related compounds in both drug substance and drug product.

The UPLC-developed methodology can demonstrate superior selectivity, sensitivity and reproducibility with significantly shortened run time and smaller usage of expensive reagent.  In addition, a UPLC/MS/MS method can be developed to quantify the oligonucleotide under evaluation in plasma and tissue for different animal speciesand will be more accurate, precise, and selective and will have a broader calibration range in plasma samples.