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Development of orthogonal chromatographic methods for the purity analysis of therapeutic oligonucleotides
Morgane Lescut, Claire Butré, Arnaud DelobelQuality Assistance,Technoparc de Thudinie 2, B-6536 Donstiennes (BELGIUM)
Oligonucleotides have been in clinical development for the past 30 years as candidate treatments for rare or genetic diseases. As of October 2020, ten of these molecules provided clear clinical benefit in rigorously controlled trials [1]. Therapeutic oligonucleotides are short chains from 20 to 35 nucleotides. These nucleotides consist of three molecules linked by covalent bonds: a nitrogen base, a sugar and a phosphate group. Therapeutic oligonucleotides can also be subtly modified to increase resistance against enzymatic digestion (Figure 1). Modifications on the phosphate and ribose are the most common. Typically, in the pharmaceutical industry, the oligonucleotides are phosphorothioate where a sulphur a tom replaces a non-bridging oxygen on the phosphate backbone. Another type of modification consists in adding an alkyl group (methyl, methoxyethyl, etc) in position 2’ of the ribose. This type of modification allows for a better lifetime and a decrease in toxicity. Oligonucleotides are synthetised by a chemical process. Despite extensive purification steps, it is impossible to reach total purity: an oligonucleotide sample does not only contain the oligonucleotide of interest of a given length n but also oligonucleotides of unwanted length, such as n-1 oligonucleotides (i.e. lacking one nucleotide). In order to determine the exact composition of an oligonucleotide sample, three types of high-performance liquid chromatography (HPLC) methods were evaluated: reversed-phase chromatography with ion pairing (IP-RP), hydrophilic interaction liquid chromatography (HILIC) and anion-exchange chromatography (AEX).[1] Vanhinsbergh CJ (2020) Analytical separation methods for therapeutic oligonucleotides, LCGC Europe 33:20-26.
CONCLUSIONThree techniques of liquid chromatography (IP-RP, AEX and HILIC) were developed for studying phosphorothioate oligonucleotides. Among these three methods, reversed-phase liquid chromatography with ion-pairing provided better results than the two others. Indeed, for IP-RP, a better separation of the oligonucleotides of different lengths were reached. It was shown that IP-RP mobile phase choice depends on the presence of a modification on the ribose moiety of the phosphorothioate oligonucleotide. Moreover, RP can be easily coupled with mass spectrometry for the identification of the different compounds, or for specific quantification of co-eluting species. The use of HILIC and AEX could be beneficial as orthogonal methods, or if IP-RP does not provide satisfactory results on particular samples.
Ion pairing agents are commonly used for the study of oligonucleotides in RP-LC as oligonucleotides are highly polar molecules and not well retained on RP stationary phases.Several parameters were tested and optimised:
• Ion pairing agents: triethylamine (TEA) with hexafluoroisopropanol (HFIP), tributylammonium acetate (TBuAA) with ethylenediaminetetraacetic acid (EDTA), and hexylamineacetate (HAA)
• UPLC columns: BEH C18, OST C18, AdvanceBio Oligonucleotides, Oligo XT, Infinity Lab Poroshell HPH
• HPLC parameters: flow rate and column temperatureConclusions:
• Waters OST BEH C18 (100x2.1 mm, 2.7 µm) column gave the best results. • HAA gave the poorest results and was excluded from the tests. • TEA/HFIP and TBuAA/EDTA were further evaluated by testing the elution for several
oligonucleotides, containing different modifications. During the optimisation of the analytical conditions it became obvious that the type of modification on the oligonucleotide played a role on the choice of the mobile phase system:
• Mobile phases containing TBuAA/EDTA gave the best results for phosphorothioate oligonucleotides with modifications on the ribose moiety.
• Mobile phase containing TEA/HFIP are better suited for phosphorothioate oligonucleotides with no modification on the ribose (Figure 2).
Figure 1: Structure of phophorothioate oligonucleotides with examples of modifications
Mobile phaseA: 10 % ACN, 5 mM TBuAA, 1 µM EDTAB: 80 % ACN, 5 mM TBuAA, 1 µM EDTA
Gradient of mobile phase
Time (min) % Mobile phase A % Mobile phase B
0 60 401 60 40
16 10 9018 10 9019 60 4025 60 40
HPLC column OST C18: 2.1 mm x 100 mm ; 1.7 µm (Waters)Flow rate 0.30 mL/minColumn temperature 60°C
Injection volume 5 µL of 0.1 mg/mL in eluent A
Detection 260 nm
Table 1: Optimal parameters in IP-RP for phosphorothioate oligonucleotides with a modification on the ribose moeity
Figure 2: Chromatogram comparison in function of mobile phases and chemical modifications of oligonucleotides
The third method used in this study was anion exchange chromatography, commonly used for “regular” oligonucleotides. However, the conditions used for “regular” oligonucleotides were not directly applicable to phosphorothioate oligonucleotides, which require mobile phases containing much more salt to be eluted from the column. Several tests were performed for optimisation of the elution on a single type of column (i.e. DNAPac):
• Two types of mobile phases were compared: sodium perchlorate buffer and a phosphate buffer with the addition of sodium bromide. The latter was selected and parameters such as phosphate concentration and gradient were studied.
• Improved chromatographic resolution was observed with increased phosphate concentration while a decrease in gradient slope led to a decrease in chromatographic resolution.
The optimised parameters (Table 3) were applied to the R&D-grade Nusinersen oligonucleotide (Figure 4). Based on the MS identification performed for IP-RP, it is assumed that the main peak is preceded by peaks of truncated n-1 and n-2 oligonucleotides.
Mobile phase
A: 90/10 H2O/ACN + 15 mM Ammonium acetate (pH 5.5)B: 10/90 H2O/ACN + 15 mM Ammonium acetate (pH 5.5)
Gradient of mobile phase
Time (min) % Mobile phase A % Mobile phase B
0 15 8532 50 5038 15 8553 15 85
HPLC column
Poroshell 120: 2.1 mm x 100 mm x 2.7 µm (Agilent)
Flow rate 0.15 mL/min
Column temperature
50°C
Injection volume
2 µL of 0.1 mg/mL in H2O/ACN (50/50)
Detection 260 nm
Table 2: Optimal parameters in HILIC
Figure 3: UV260 chromatogram of Nusinersen in HILIC
Until now, only a limited number of studies have reported the separation of oligonucleotides by liquid chromatography with hydrophilic interactions. The developments performed in this study are based on a publication using ammonium acetate for elution [2]. The influence of a large number of chromatographic parameters was evaluated on the overall quality of the elution and the peak resolution:
• The mobile phase composition as well as the gradient• The sample preparation and the injection volume • The influence of the column temperature and the flow rate
Optimal parameters were defined (Table 2) for which an acceptable separation of the different species was reached. As often with HILIC separation, the sample preparation is an important parameter and only a limited volume was injected for proper chromatographic quality. It is assumed that oligonucleotides with a respective deficit of 1 to 3 nucleotides elute before the oligonucleotide of interest of a given length n, in the example of the R&D-grade Nusinersen oligonucleotide (Figure 3).
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Based on MS identification using an ESI-QTOF system hyphenated to the LC, it was determined that peaks eluting before the main peak are truncated oligonucleotides. In the example of the oligonucleotide Nusinersen, truncated oligonucleotides missing from 1 to 5 nucleotides were identified as well as an oligonucleotide with an additional nucleotide (n+1) eluting after the main oligonucleotide.
Figure 4: UV260 chromatogram of Nusinersen in AEX
Mobile phase
A: 10 % ACN, 50 mM Na3PO4 pH 11.7B: 10 % ACN, 50 mM Na3PO4 pH 11.7 + 2 M NaBr
Gradient of mobile phase
Time (min) % Mobile phase A % Mobile phase B
0 100 01 100 0
21 0 10026 0 10031 100 032 100 0
HPLC column
DNAPac: 4 mm x 250 mm x 8 µm (Thermo Fisher)
Flow rate 1 mL/min
Column temperature
35°C
Injection volume
10 µL of 0.1 mg/mL in H2O
Detection 260 nm
Table 3: Optimal parameters in AEX
INTRODUCTION
IP-RP
HILIC AEX
[2] Lobue PA, Jora M, Addepalli B, Limbach PA (2019) Oligonucleotide analysis by hydrophilic interaction liquid chromatography-mass spectrometry in the absence of ion-pair reagents, J. Chromatogr A, 1595:39-48
