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Generic anion-exchange chromatography method for analytical and preparative separation of nucleotides in the development and manufacture of drug substances
Accepted Manuscript Title: Generic anion-exchange chromatography method for analytical and preparative separation of nucleotides in the development and manufacture of drug substances Authors: Fuh-Rong Tsay, Imad A. Haidar Ahmad, Derek Henderson, Nicole Schiavone, Zhijian Liu, Alexey A. Makarov, Ian Mangion, Erik L. Regalado PII: DOI: Reference:
S0021-9673(18)31525-5 https://doi.org/10.1016/j.chroma.2018.12.018 CHROMA 359879
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
31 October 2018 1 December 2018 10 December 2018
Please cite this article as: Tsay F-Rong, Haidar Ahmad IA, Henderson D, Schiavone N, Liu Z, Makarov AA, Mangion I, Regalado EL, Generic anion-exchange chromatography method for analytical and preparative separation of nucleotides in the development and manufacture of drug substances, Journal of Chromatography A (2018), https://doi.org/10.1016/j.chroma.2018.12.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Generic anion-exchange chromatography method for analytical and preparative separation of nucleotides in the development and manufacture of drug substances
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Fuh-Rong Tsay, Imad A. Haidar Ahmad, Derek Henderson, Nicole Schiavone, Zhijian Liu, Alexey A. Makarov, Ian Mangion, Erik L. Regalado*
Process Research and Development, MRL, Merck & Co., Inc., Rahway, NJ 07065, USA Corresponding author. Tel.: +1 732 594 5452 (E.L.R)
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*E-mail addresses: [email protected]
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Generic ion-exchange method for analytical and preparative separation of nucleotides Chromatographic resolution of over 20 nucleotides in a single experimental run The use of a cost-effective resin available in bulk format for preparative separation is demonstrated Aqueous ammonium bicarbonate-based eluent profile allows convenient drying process Fully implemented for preparative separation of nucleotides in multicomponent reaction mixtures
ABSTRACT:
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Highlights
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Nucleotides are among the most frequently used chemical building blocks in the research, development and manufacture of drug substances. They are composed of three highly
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polar subunit molecules (a nucleobase, a sugar, and at least one phosphate group), which makes their separation and analysis very challenging by conventional liquid
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chromatography techniques. Herein, we describe a simple, efficient, and cost-effective ion-exchange chromatography (IEC) method for the separation and purification of over 20 nucleotides. This method combines the use of a Tosoh TSKgel SuperQ-5PW resin in conjunction with a fully aqueous eluent profile (ammonium bicarbonate-based) that allows for a straightforward scale-up transition and convenient drying process with minimal environmental impact. This generic method was optimized using 1
chromatography simulation software (ACD Labs/LC Simulator) and successfully applied to the preparative purification of multicomponent nucleotide mixtures using readily available Fast Protein Liquid Chromatography (FPLC) instrumentation. These IEC method conditions can be effectively applied as the starting point for method development and isolation of other highly polar nucleotide species beyond those
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investigated in this study.
Keywords: ion chromatography; method development; preparative separation; nucleotides; chromatographic modeling and simulation; generic method. 1. Introduction
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In recent years, the development and implementation of generic or more universal
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chromatographic methods capable of baseline resolution of multiple targeted analytes in a
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single experimental run has proliferated in both academia and industry. Highly efficient standardized methods that cover a broad variety of analytes and chemical groups
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commonly used or obtained during the development and manufacture of drug substances have been recently reported, e.g. the analysis of solvents and volatile amines by Gas
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Chromatography (GC) [1,2], dehalogenation impurities and mixtures of halogen isomers
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[3-6], drug metabolites and analogs [7-12], direct analysis of chiral active pharmaceutical ingredients (APIs) and their counterions [13] by High-Performance Liquid
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Chromatography (HPLC), enantiopurity analysis across an entire synthetic route using a single chiral Coreshell column [14], generic chiral method development in supercritical fluid chromatography (SFC) [15], and synthetic intermediates containing multiple
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stereocenters by 2D- LC [16], amongst others [1,2,17-25]. The increased implementation of generic chromatographic methods in the pharmaceutical industry has been enabled by
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the evolution of improved stationary phases, instrumentation, and chromatographic particle technology [26-32]. “On demand” chromatographic method development and optimization is the primary approach in some areas of research and development. However, in other areas where the pace of research is fast and more dynamic (e.g. medicinal chemistry, process optimization, amongst other areas of pharmaceutical research and development) [16,332
36], generic chromatographic methods are often employed as a time-saving expedient [25], with the purpose of minimizing the time spent developing new analytical methods prior to each analysis session. This generic or more universal approach serves to cover a broad spectrum of compound classes, but can be also used as the starting point during method development for the analysis of other related analytes beyond the initial target
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[2].
Previously published generic methods are directed towards quantitative assays, reaction monitoring, impurity mapping and other analytical applications. However, this concept has not yet been applied to scale-up separation processes, where turnaround time, costsavings and method “greenness” are of paramount importance in the delivery of highly pure pharmaceutical drugs and synthetic intermediates. Large scale purification
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laboratories are typically a very expensive commodity, where method development and
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optimization are significantly more costly than their analytical counterparts. Separation of
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complex mixtures of closely related species can already be challenging even using stateof-the-art UHPLC on sub-2μm particle stationary phases or Coreshell column
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technologies. Handling these mixtures at preparative scale is more problematic since UHPLC columns and instrumentation are not suited for purification purposes. In addition
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to the aforementioned limitations, scale-up separations encompass additional challenges
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including sample solubility, loading optimization, fraction collection, waste accumulation and disposal; and equally important, the drying process. In this regard, developing a
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chromatographic method that combines the use of a cost-effective stationary phase (available in bulk format) and an easily removed mobile phase eluent without the need of
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a desalting requisite prior to drying, makes the difference between success or failure in the pursuit of a feasible scale up purification. Dealing with chromatographic analysis of nucleobases, sugars or compounds containing
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phosphate groups can be very challenging due to the strong polarity of these species. Separation and analysis of nucleotides (compounds containing all of the aforementioned molecular subunits) can be nightmarish to even the most skilled chromatographers. In many instances, chromatographic resolution of multicomponent nucleotide mixtures have been identified as an extremely challenging task, requiring the use of “non-conventional”
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liquid chromatography approaches in order to achieve acceptable retention, peak shape, and selectivity, e.g. hydrophilic interaction liquid chromatography (HILIC), enhancedfluidity liquid chromatography (EFLC), amongst others [37-42]. Most of the prior efforts at separating nucleotide mixtures focused on a very narrow group of nucleotides at analytical scale and do not have the potential for use in a more generic way when
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working with the diverse group nucleotides commonly found in pharmaceutical research and development. More importantly, these analytical scale separations cannot be successfully translated to large scale separations.
In this study, a novel strong anion-exchange chromatography procedure that meets all of the key requisites for the separation of over 20 of the most common nucleotides used as starting materials and synthetic intermediates in PR&D is reported. The use of this
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simple, efficient and cost-effective generic IEC method for the purification of
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multicomponent nucleotide mixtures is demonstrated with scale up data provided to
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enable its application in both analytical and large scale separation laboratories.
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2. Experimental
2.1. Instrumentation
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Anion-exchange chromatography screening and optimization experiments were
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performed on an Agilent 1260 system (Agilent Technologies, Palo Alto, CA, USA). The Agilent system was comprised of a G1311B quaternary pump, a G1329B ALS
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autosampler, a G1330B thermostat, a G4212B diode array detector, a G1316C 1260 column compartment, a G1364C 1260 FC-AS fraction collector, and a G1330B
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thermostat. Anion-exchange scale up demonstration and purification experiments were performed on both an Agilent semi-prep 1200 system (Agilent Technologies, Palo Alto, CA, USA) and an AKTA Pilot preparative system. The Agilent system was comprised of
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two G1361A prep pumps, a G2258A ALS autosampler, a G1315D diode array detector, and two G1354B Prep FC fraction collectors. The AKTA Pilot (GE Healthcare Life Sciences, Marlborough, MA, USA) was self-contained system with both UV and conductivity detection. The AKTA system was controlled by GE Healthcare Life Sciences UNICORN software, version 5.31 (GE Healthcare Life Sciences), a G1367A
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WPALS autosampler, a G1316A column compartment, and a G1315B diode array detector. 2.2 Chemicals and reagents The names and the structures of the compounds used in this study are illustrated in Figure
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1. Compounds 1-8 were obtained in house. Compounds 9-20 were obtained from SigmaAldrich, Inc. (St Louis, MO, USA). 2.3. Preparation of buffer solutions
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1 Molar (1 M) ammonium bicarbonate (NH4HCO3) in water (H2O); solution pH = 7.9: 79.06 grams (g) of NH4HCO3 were dissolved in 1 liter (L) of Millipore HPLC grade water. Solution is degassed for 20 min (vacuum, 27 torr).
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20 mM Tris Buffer in H2O solution, pH = 8: 2.42 g of Tris Base (tris-(hydroxymethyl)aminomethane, MW = 121.14 g/mole), were dissolved in 1L Millipore HPLC grade
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water.
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20 mM Tris, 0.5 M NaCl Buffer in H2O solution, pH = 8: 2.42 g of Tris Base (tris(hydroxymethyl)-aminomethane, MW = 121.14 g/mole) and 29.22 g NaCl were
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dissolved in 1L Millipore HPLC grade water.
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1 M ammonium acetate (CH3CO2NH4) in H2O solution, pH=6.88: 77 g of ammonium acetate, (MW = 77.08 g/mole) were dissolved in 1L Millipore HPLC grade water. 20 mM phosphate buffer in H2O solution, pH = 7.5: 2.31 g of Na2HPO4 (sodium
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phosphate, dibasic, (MW = 141.96 g/mole) and 0.52 g of NaH2PO4-H2O (sodium phosphate, mono basic, mono hydrate, MW = 137.99 g/mole) were dissolved in 1L
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Millipore HPLC grade water. 20 mM phosphate buffer, 1 M NaCl in H2O solution, pH = 7.5: 2.31 g of Na2HPO4 (sodium phosphate, dibasic, MW = 141.96 g/mole), 0.52 g of
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NaH2PO4-H2O (sodium phosphate, mono basic, mono hydrate, MW = 137.99 g/mole), and 58.44 g of NaCl were dissolved in 1L Millipore HPLC grade water. 20 mM phosphate buffer in water (pH 8.5) solution: 2.78 g of Na2HPO4 dibasic (MW 141.96 g/mole) and 0.06 g of NaH2PO4-H2O mono (MW137.99 g/mole) were dissolved in 1L Millipore water. 20 mM phosphate buffer in water (pH 8.5) solution with 1 M
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NaCl: 2.78 g of Na2HPO4 dibasic (MW 141.96 g/mole), 0.06 g of NaH2PO4-H2O mono (MW137.99 g/mole), and 58.44 g of NaCl were dissolved in 1L Millipore water. 2.4. Screening for stationary phases and mobile phases Chromatographic column screening was performed to determine optimal
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chromatographic conditions for preparative ion-exchange chromatography (IEC).
Initially, two anion-exchange (AEX) columns were screened in conjunction with three
separate mobile phase combinations. The AEX columns selected for the screening were the Proteomix SAX-NP10 (4.6 x 50 mm) and the WAX-10NP (4.6 x 50 mm), both
purchased from Sepax. The first mobile phase examined consisted of eluents (A) 20 mM Tris buffer (pH 8.0) and (B) 20 mM Tris, 0.5 M NaCl buffer, (pH 8.0). A gradient
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elution profile (30 minutes) was executed as follows: 0 min (0% B), 20 min (100% B),
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20.1-30 min (0% B). The second mobile phase examined consisted of eluents (A) water and (B) 1 M ammonium acetate in water (pH 6.7) and the third mobile phase examined
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was (A) water and (B) 1M ammonium bicarbonate in water (pH 8.01). Gradient elution
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profiles (45 minutes) for the second and third mobile phases were executed as follows: 02 min (1% B), 2-22 min (100% B), 22.5-45 min (1% B). All screens consisted of 10 µL
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injections performed in duplicate with a flow rate of 0.5 mL/min, column temperature of
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25 °C, and UV detection at 260 nm (reference signal at 360 nm). The next round of column screening was carried out with the following strong anionexchange (SAX) columns; the UniCore 10Q, AEX column (4.6 x 150 mm, 10 µm
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particles) purchased from Nexgen and the TSKgel SuperQ 5PW (10) (4.6 x 150 mm, 10 µm particles) purchased from TOSOH. As before, three mobile phase combinations were
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applied with these columns. The eluents employed for the screening were as follows: Eluent 1 - (A) 20 mM phosphate buffer (pH 8.5), (B) 20 mM phosphate buffer (pH 8.5),
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1 M NaCl; Eluent 2 – (A) Water, (B) 1 M ammonium bicarbonate (pH 8.0); Eluent 3 – (A) 20 mM phosphate buffer (pH 7.5), (B) 20 mM phosphate buffer (pH 7.5), 1 M NaCl. The gradient elution profiles for all three mobile phases were executed as follows: 0 min (5% B), 80-100 min (100% B), 100.1-120 min (5% B). All of these column screens were conducted at a flow rate of 0.27 mL/min, column and sample temperatures of 22°C and 45°C, respectively, and UV detection set to 210, 254, and 280 nm. 6
2.5. Experimental conditions used for LC-Simulator modeling Upon review and evaluation of the afore-listed screening study, the following column and chromatographic conditions were selected for continued IEC method development for separation of 20 nucleotides (Fig. 1) including chromatographic modeling and simulation
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(see some chromatographic profiles in Fig. 2a): IEC resin: TOSOH TSKgel SuperQ 5PW (4.6 mm x 150 mm, 10 µm particles) column by a gradient elution using eluent A: water
and eluent B: 1 M ammonium bicarbonate (pH 8.0). Flow rate: 0.27 mL/min. Two eluent gradients, 10-90% B in 40 min and 10-90% B in 80 min, each followed by 20 min re-
equilibration, were executed at three temperatures (30, 40, and 60 °C) in order to build a 3D resolution map. The resultant experimental data was input and processed using
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ACD/LC Simulator 2015 Release (Version L10R41), Advanced Chemistry Development,
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Inc. (ACD), Toronto, Ontario, Canada, further referred to as LC Simulator.
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2.6. Optimized Anion-Exchange Chromatography method
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The 3D resolution map was built based on the conditions listed-above, which allowed us to develop the following optimized method for separation of all 20 nucleotides using the same IEC resin and mobile phase. Gradient: 10% to 90% B in 60 min, with 20 min re-
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equilibration time at 10% B. Flow rate: 0.27 mL/min. Column temperature: 30°C.
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Injection volume: 5 μL. UV detection: 254 nm. Experimental chromatogram is shown in Fig. 2b.
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2.7. Chromatographic conditions for isolation and purification of synthetic intermediates
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and process related impurities IEC scale-up purification was implemented on a 70 mm × 500 mm (20 μm particles), TOSOH TSKgel SuperQ 5PW (20) column via gradient elution chromatography (AKTA
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Pilot) derived from the best conditions introduced above. Purification of intermediate #3: a gradient elution profile (at flow rate of 80 mL/min) was performed in which the eluent delivered was 0-32 min (30% B), 60-100 min (40% B), 100.1-120 min (100% B), 120.1150 min (30% B). Mobile phase component (A) was HPLC grade water and mobile phase component (B) was 1 M ammonium bicarbonate (pH 8.0). Sample load volume was 250 mL (4g mixture of active 25 wt% intermediate #3 in water) and the IEC column 7
and sample feed were maintained at ambient (22°C) temperature, respectively. Purification of intermediate #7: a gradient elution profile (at flow rate of 80 mL/min) was performed in which the aforementioned mobile phase delivered was 0-35 min (35% B), 60-100 min (45% B), 100.1-125 min (100% B), 125.1-155 min (35% B). In likewise fashion, the sample load volume was 250 mL (3.7 g mixture of active 27 wt%
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intermediate #7 in water) under ambient (22°C) column and sample feed temperatures, respectively.
A smaller scale IEC purification was carried out on a 35 mm × 250 mm (20 μm particles), TOSOH TSKgel SuperQ 5PW (20 μm) column via gradient elution
chromatography (Agilent 1200 series) under the aforementioned method conditions.
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Purification of process related impurity #4: a gradient elution profile (at flow rate of 20 mL/min) was performed in which the mobile phase (mobile phase (A) was water and
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mobile phase (B) was 1 M ammonium bicarbonate (pH 8.0)) delivered was 0-16 min
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(30% B), 40-60 min (40% B), 60.1-70 min (100% B). The sample load volume was 20
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mL (50 mg of desired material #4 in water) with an ambient temperature of 22°C for the column and sample feed, respectively. Purification of process related impurity #8: a
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gradient elution profile (at flow rate of 20 mL/min) was performed in which the mobile phase (mobile phase (A) was water and mobile phase (B) was 1 M ammonium
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bicarbonate (pH 8.0) delivered was 0-42 min (35% B), 60 min (42% B), 60.1-70 min (100% B). The sample load volume was 14 mL (40 mg of desired material #8 in water)
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with an ambient temperature of 22 °C for the column and sample feed, respectively.
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3. Results and Discussion Separation and analysis of multicomponent mixtures of nucleotides is never easy, and requires chromatographic approaches beyond conventional reversed phase liquid
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chromatography (RPLC). Other well-established techniques in the pharmaceutical industry such as normal phase liquid chromatography (NPLC) and supercritical fluid chromatography (SFC) are often inapplicable due to exceedingly strong retention, aggravated tailing effects, and poor chromatographic performance. The development and optimization of a generic chromatographic method for the separation and purification of multiple nucleotides in a single experimental run is essential for a fast and reliable 8
turnaround of analytical results and batch purification deliveries. All of the 20 nucleotides selected in this investigation for method development form part of a comprehensive list of starting materials and intermediates that have been used by synthetic and process chemists in our laboratories for pharmaceutical research and development. The list also includes dinucleotides and other structurally diverse
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nucleotides containing different nucleobases and number of phosphate groups (Figure 1). We examined each of these mixtures using several chromatographic screening systems that are routinely used within these laboratories for the development of analytical
methods[4,43,44]. As expected, RPLC and SFC showed very poor chromatographic
performance and inadequate resolution. IEC screening, by contrast, provided conditions for the separation of multiple nucleotides shown in figure 1. A complete listing of the
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columns, eluents, and methods used in the screening experiments is described in the
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Experimental Section. It is important to point out that selection of the best screening hits
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for method development is based on criteria that enable both analytical and large scale separations, e.g. peak shape, retention, separation, resin cost and availability in bulk
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format; and equally important, a mobile phase that can be directly removed by conventional drying technologies. In this regard, TOSOH TSKgel SuperQ-5PW resin
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proved to be an excellent stationary phase candidate for method development and
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optimization. These columns are packed with porous hydrophilic polymer (polymethacrylate based) beads which are modified with a strong anion-exchange group
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(trimethyl-amino ligands). TOSOH TSKgel SuperQ-5PW resin shows excellent chromatographic performance for the separation of all 20 nucleotides when combined
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with a fully aqueous ammonium bicarbonate-based eluent. The use of organic eluents in the mobile phase is not required which serves to reduce cost and environmental impact,
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something of vital importance in the implementation of large scale purification processes. The aforementioned screening results were subsequently optimized in order to develop a method that separates all of the 20 nucleotides in a single experimental run. Such generic chromatographic conditions could be effectively applied for the isolation and purification of nucleotide species beyond the scope of this list, where reduction in method development efforts can lead to significant time savings when multiplied across the many 9
samples submitted for purification. The results outlined in Figure 2a illustrate initial attempts at the separation of the entire mixture. Compounds were subdivided into five different mixtures to minimize sample degradation and simplify peak identification. The resultant experimental data was input and processed using ACD/LC Simulator software. Two eluent gradients: 10-90%B in 40 min and 10-90%B in 80 min, each followed by 20
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min re-equilibration, were run at three different temperatures (30, 40, and 60 °C) in order to construct a 3D resolution map that was subsequently used for the prediction of chromatographic conditions and elution profiles depicted in figure 2b.
The 3D resolution map shows a very small and narrow area (orange) of robust conditions allowing for the efficient and convenient resolution of all 20 nucleotides using a linear
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gradient elution, in particular, all of the critical pairs that are very difficult to separate (e.g. nucleotides 2 and 5, 3 and 6) using the initial gradient and column temperature.
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Figure 2b also shows the overlaid chromatographic profiles from both simulated and
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experimental data illustrating the power of modern chromatography simulation and
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modeling software when applied for method development. Interestingly, some critical pair separations exhibited elution order variations in going from initial screening to the
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optimized conditions. As is expected for IEC, all of the dinucleotides are more retained, with increased overall retention in concert with the number of phosphate groups. These
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simulated chromatographic data were compared to experimental outcomes. The comparisons outlined in table 1 show an excellent match, with overall ∆tR differences
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below 0.26 min between predicted and experimental data. Following suit, the aforementioned analytical IEC conditions (resin and buffer eluent)
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were effectively used as the starting point during method development for the isolation and purification of nucleotide intermediates and process related impurities. This approach
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serves to streamline scale up transition and speed up purification turnarounds, which leads to significant time savings when multiplied across many pipeline programs. Figure 3 shows an impressive collection of highly efficient separations across a challenging set of closely related nucleotides in reaction mixtures. All of the optimizations were performed by minimal modification of the mobile phase gradient, column dimensions and flow rate while retaining the same IEC resin and ammonium bicarbonate based eluent. 10
A baseline separation of intermediate #3 from a very complex reaction mixture (Fig. 3a) was achieved on large scale using readily available preparative FPLC instrumentation (AKTA Pilot) on a 70 mm I.D. x 500 mm length (20 μm) column. This method allowed us to process 4g of crude per injection enabling the delivery of 12g of intermediate #3 (96% purity) from a very complex reaction mixture. It is important to point out that the
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same method was applied to the isolation of AMP and ADP (nucleotide #1 and #2) which are closely related process byproducts formed in the reaction. Figure 3b illustrates
another successful scale up purification easily accomplished by minor modification of gradient conditions using the same column and buffer. This method was effectively
implemented to obtain 34g of intermediate #7 with 99% purity, an excellent result from another complex mixture requiring no further purification. Interestingly, this
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chromatographic procedure is also very effective for the separation of other nucleotides
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formed in the same reaction (e.g. compounds #5 and #6). In another example depicted in figure 3c, a similar approach using readily available semipreparative HPLC
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instrumentation ((Agilent 1200 series) on a 35 mm I.D. x 250 mm length (20 μm)
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column) was applied to the isolation of a process related impurity for use as reference standard (compound # 4: 60 mg isolated with 98% purity). Note that the same IEC
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conditions can be used for separation of compound # 3 which is also a reaction product.
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The final example shown in figure 3d is related to the isolation of another impurity (compound #8) for reference standard purposes. The same column with a modified step gradient was utilized to deliver 50 mg of this impurity (99% purity). It is important to
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highlight that all of these rich cut fractions contain the desired analytes in a fully aqueous ammonium bicarbonate buffer and can be directly dried by lyophilization without the
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need for a desalting procedure after purification. This is a critical feature that makes these large scale separation methods so effective in accelerating purification deliveries and
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minimizing chemical degradation. In addition, the use of organic solvents such as acetonitrile and alcohols are not required which serves to reduce toxic waste and minimize environmental impact.
4. Conclusions 11
Pharmaceutical, agricultural, biomedical, and other research and development disciplines, often involve the extensive use of nucleotides as building blocks for synthetic route development. Consequently, chemists have to spend a considerable amount of time and resources developing analytical and preparative chromatographic methods prior to each purification session. To better meet these needs, a simple, efficient, and cost-effective
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IEC method for separation and purification of over 20 nucleotides was developed and implemented. Our method combines the use of a Tosoh TSKgel SuperQ-5PW in
conjunction with a fully aqueous eluent profile (ammonium bicarbonate-based) that
enables a straightforward scale up transition and convenient drying process with minimal environmental impact. This generic method was optimized using ACD Labs
chromatography simulation software and successfully applied to the large scale
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purification of complex mixtures of nucleotides by using readily available preparative
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FPLC instrumentation. In addition, these IEC conditions combined with ACD Labs modeling can be effectively used as a starting point during method development for the
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isolation of other highly polar nucleotide species beyond the list described in this study. Acknowledgements
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We thank Dr. Mirlinda Biba (Merck & Co, Inc.) and collaborators at Sepax Technologies for fruitful discussions.
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F.T. Mattrey, A.A. Makarov, E.L. Regalado, F. Bernardoni, M. Figus, M.B. Hicks, J. Zheng, L. Wang, W. Schafer, V. Antonucci, S.E. Hamilton, K. Zawatzky, C.J. Welch, Current challenges and future prospects in chromatographic method development for pharmaceutical research, Trends Anal Chem 95 (2017) 36.
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V. Hoguet, J. Charton, P.-E. Hecquet, C. Lakhmi, E. Lipka, Supercritical fluid chromatography versus high performance liquid chromatography for enantiomeric and diastereoisomeric separations on coated polysaccharides-based stationary phases: Application to dihydropyridone derivatives, J Chromatogr A 1549 (2018) 39.
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S. Lin, S. Dikler, W.D. Blincoe, R.D. Ferguson, R.P. Sheridan, Z. Peng, D.V. Conway, K. Zawatzky, H. Wang, T. Cernak, I.W. Davies, D.A. DiRocco, H. Sheng, C.J. Welch, S.D. Dreher, Mapping the dark space of chemical reactions with extended nanomole synthesis and MALDI-TOF MS, Science (2018) in press.
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X.W. Diefenbach, I. Farasat, E.D. Guetschow, C.J. Welch, R.T. Kennedy, S. Sun, J.C. Moore, Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry, ACS Omega 3 (2018) 1498.
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J. Bostrom, D.G. Brown, R.J. Young, G.M. Keseru, Expanding the medicinal chemistry synthetic toolbox, Nature Reviews Drug Discovery (2018).
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M.C. Beilke, M.J. Beres, S.V. Olesik, Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A "green" technique, J Chromatogr A 1436 (2016) 84.
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G.S. Philibert, S.V. Olesik, Characterization of enhanced-fluidity liquid hydrophilic interaction chromatography for the separation of nucleosides and nucleotides, J Chromatogr A 1218 (2011) 8222.
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K. Inoue, R. Obara, T. Hino, H. Oka, Development and Application of an HILICMS/MS Method for the Quantitation of Nucleotides in Infant Formula, J Agric Food Chem 58 (2010) 9918.
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N.L.T. Padivitage, M.K. Dissanayake, D.W. Armstrong, Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column, Analytical and Bioanalytical Chemistry 405 (2013) 8837.
[41]
D. Garcia-Gomez, E. Rodriguez-Gonzalo, R. Carabias-Martinez, Stationary phases for separation of nucleosides and nucleotides by hydrophilic interaction liquid chromatography, Trends Anal Chem 47 (2013) 111.
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E. Johnsen, S.R. Wilson, I. Odsbu, A. Krapp, H. Malerod, K. Skarstad, E. Lundanes, Hydrophilic interaction chromatography of nucleoside triphosphates with temperature as a separation parameter, J Chromatogr A 1218 (2011) 5981.
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E.L. Regalado, W. Schafer, R. McClain, C.J. Welch, Chromatographic resolution of closely related species: Separation of warfarin and hydroxylated isomers, J Chromatogr A 1314 (2013) 266.
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[34]
W. Schafer, T. Chandrasekaran, Z. Pirzada, C. Zhang, X. Gong, M. Biba, E.L. Regalado, C.J. Welch, Improved Chiral SFC Screening for Analytical Method Development, Chirality 25 (2013) 799.
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[44]
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Fig. 1. Structures and names of nucleotides used in this study
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Fig. 2. Method development for separation of 20 nucleotides from modeling (a) to experimental conditions (b). All chromatographic conditions applied to build the modeling and 3D resolution map are described in the Experimental section, as well as the final optimized conditions.
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Fig. 3. Purification of nucleotides intermediates and impurities used/formed during the synthesis of more complex molecules at MSD Research Laboratories. IEC resin: TSKgel SuperQ-5PW (20 μm), eluent A: H2O, Eluent B: 1 M ammonium bicarbonate, temperature: ambient, detection: 254 nm. Injection volume and Gradient elution as follow: a) 70 mm I.D. x 500 mm length column; 16 mg/mL, 250 mL injected; 30 to 40 % eluent B in 100 min. b) 70 x 500 mm column; 14.8 mg/mL, 250 mL injected; 35 to 45 % eluent B in 100 min. c) 35 x 250 mm column; 5 mg/mL, 20 mL injected; 30 to 40 % eluent B in 60 min. d) 35 x 250 mm column; 4.0 mg/mL, 14 mL injected; 35 to 42 % eluent B in 60 min, then go to 100% B in 0.1 min, with a final 10 min hold. Chromatographic conditions are outlined in more detail in the Experimental section.
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Fig. 1.
Peak #
Compound name 2‘R-adenosine-5-(1-R-momonophosphate)
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1 2
2‘R-adenosine-5-(1-R-diphosphate)
3
2‘R-adenosine-5-(1-R-triphosphate)
4
2‘R-adenosine-5-(1-R-tetraphosphate)
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3‘R-guanosine-5'-R-monophosphate
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3‘R-guanosine-5'-R-diphosphate
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3‘R-guanosine-5'-R-triphosphate
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3‘R-guanosine-5'-R-tetraphosphate
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Adenosine 3′,5′-cyclic monophosphate sodium salt
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Adenosine 5′-monophosphate sodium
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Adenosine 5′-diphosphate sodium
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Adenosine 5′-triphosphate disodium salt
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Guanosine 5′-diphosphate sodium salt
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Guanosine 5′-triphosphate sodium salt hydrate
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β-Nicotinamide adenine dinucleotide sodium salt β-Nicotinamide adenine dinucleotide phosphate sodium salt hydrate
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Nicotinamide guanine dinucleotide sodium salt
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Nicotinamide hypoxanthine dinucleotide sodium salt
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Guanosine 3′,5′-cyclic monophosphate sodium salt Guanosine 5′-monophosphate disodium salt hydrate
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Fig. 2.
2a) Structure of stationary phase and experiments to build the model 12
10 11
1500
15
16
14
experimental
1000
Strong anion exchanger phase (note: R’=propriety)
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19 20
0 5
15
70
0.00010
17 17
16 16 44 33
0.00006
19 19
22
14 14
0.00005
88 77
66 55
11
99
0.00004
55
65
1.50
55
1.43 1.36 1.29 1.21 1.14 1.07 1.00
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0.93 0.86
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0.79 0.71
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45
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15 15
Column Temperature, °C
20 20
0.00007
35
1.57
60
12 12
10 10
Mix 5
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Mix 4 Mix 2 Mix 3 Mix 1
1.64
65
18 18
0.00008
8
2
1.72
Predicted Experimental
0.00009
7
18
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2b) Optimum conditions
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5
1
500
4
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0.00003
0.64
40
0.00002
0.57 0.50 0.43
35
15
20
25
30
35
40
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50
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60 min
0.36 0.29 0.21
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0.14 0.07 8
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32
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48
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96
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120 0.00
Time, min
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Fig. 3.
Resin: TSKgel SuperQ-5PW
Mobile phase: NH4HCO3 based eluent
a) Large scale purification of Merck intermediate #3
Crude: 47.5 LCAP, 25 wt% Crude: 48.5g, purified: 12.2g Final Purity: 96 LCAP
3000
Desired intermediate #3
#2
2000
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#1
1000
0 0
50
100
150
b) Large scale purification of Merck intermediate #7 Desired intermediate #7
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Crude: 75 LCAP, 27 wt% Crude: 123g, purified: 34.2g Final Purity: 99 LCAP
3000
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#5 2000
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#6
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0 0
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c) Purification of Merck impurity #4 for reference standard
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1200 Crude: 51 LCAP Purified: 60mg Final Purity: 98 LCAP
Desired imp #4
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800
#3
0
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20
30
40
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0
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400
50
60
d) Purification of Merck impurity #8 for reference standard Crude: 75 LCAP Purified: 50mg Final Purity: 99 LCAP
Desired imp #8
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mAU
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#7 500
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min
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Table 1. Summary of predicted and experimental retention time values for all 20 nucleotides
tR, predicted
tR,experimental ∆tR (%)
1 2 3 4 5 6 7 8 9 10 11 12 13
43.84 50.17 53.57 55.86 50.50 53.80 57.06 58.67 25.56 28.81 37.98 42.99 35.14
43.63 49.94 53.34 55.60 50.27 53.57 56.83 58.42 25.50 28.83 37.92 42.88 34.98
0.48 0.46 0.43 0.47 0.46 0.43 0.40 0.43 0.24 0.07 0.16 0.26 0.75
14 15 16 17 18 19 20
35.73 42.73 46.84 10.61 15.73 17.99 31.97
36.00 42.53 46.61 10.60 15.70 18.01 31.86
0.46 0.47 0.49 0.09 0.35 0.19 0.11
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S0021-9673(18)31525-5 https://doi.org/10.1016/j.chroma.2018.12.018 CHROMA 359879
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
31 October 2018 1 December 2018 10 December 2018
Please cite this article as: Tsay F-Rong, Haidar Ahmad IA, Henderson D, Schiavone N, Liu Z, Makarov AA, Mangion I, Regalado EL, Generic anion-exchange chromatography method for analytical and preparative separation of nucleotides in the development and manufacture of drug substances, Journal of Chromatography A (2018), https://doi.org/10.1016/j.chroma.2018.12.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Generic anion-exchange chromatography method for analytical and preparative separation of nucleotides in the development and manufacture of drug substances
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Fuh-Rong Tsay, Imad A. Haidar Ahmad, Derek Henderson, Nicole Schiavone, Zhijian Liu, Alexey A. Makarov, Ian Mangion, Erik L. Regalado*
Process Research and Development, MRL, Merck & Co., Inc., Rahway, NJ 07065, USA Corresponding author. Tel.: +1 732 594 5452 (E.L.R)
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*E-mail addresses: [email protected]
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Generic ion-exchange method for analytical and preparative separation of nucleotides Chromatographic resolution of over 20 nucleotides in a single experimental run The use of a cost-effective resin available in bulk format for preparative separation is demonstrated Aqueous ammonium bicarbonate-based eluent profile allows convenient drying process Fully implemented for preparative separation of nucleotides in multicomponent reaction mixtures
ABSTRACT:
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Highlights
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Nucleotides are among the most frequently used chemical building blocks in the research, development and manufacture of drug substances. They are composed of three highly
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polar subunit molecules (a nucleobase, a sugar, and at least one phosphate group), which makes their separation and analysis very challenging by conventional liquid
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chromatography techniques. Herein, we describe a simple, efficient, and cost-effective ion-exchange chromatography (IEC) method for the separation and purification of over 20 nucleotides. This method combines the use of a Tosoh TSKgel SuperQ-5PW resin in conjunction with a fully aqueous eluent profile (ammonium bicarbonate-based) that allows for a straightforward scale-up transition and convenient drying process with minimal environmental impact. This generic method was optimized using 1
chromatography simulation software (ACD Labs/LC Simulator) and successfully applied to the preparative purification of multicomponent nucleotide mixtures using readily available Fast Protein Liquid Chromatography (FPLC) instrumentation. These IEC method conditions can be effectively applied as the starting point for method development and isolation of other highly polar nucleotide species beyond those
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investigated in this study.
Keywords: ion chromatography; method development; preparative separation; nucleotides; chromatographic modeling and simulation; generic method. 1. Introduction
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In recent years, the development and implementation of generic or more universal
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chromatographic methods capable of baseline resolution of multiple targeted analytes in a
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single experimental run has proliferated in both academia and industry. Highly efficient standardized methods that cover a broad variety of analytes and chemical groups
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commonly used or obtained during the development and manufacture of drug substances have been recently reported, e.g. the analysis of solvents and volatile amines by Gas
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Chromatography (GC) [1,2], dehalogenation impurities and mixtures of halogen isomers
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[3-6], drug metabolites and analogs [7-12], direct analysis of chiral active pharmaceutical ingredients (APIs) and their counterions [13] by High-Performance Liquid
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Chromatography (HPLC), enantiopurity analysis across an entire synthetic route using a single chiral Coreshell column [14], generic chiral method development in supercritical fluid chromatography (SFC) [15], and synthetic intermediates containing multiple
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stereocenters by 2D- LC [16], amongst others [1,2,17-25]. The increased implementation of generic chromatographic methods in the pharmaceutical industry has been enabled by
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the evolution of improved stationary phases, instrumentation, and chromatographic particle technology [26-32]. “On demand” chromatographic method development and optimization is the primary approach in some areas of research and development. However, in other areas where the pace of research is fast and more dynamic (e.g. medicinal chemistry, process optimization, amongst other areas of pharmaceutical research and development) [16,332
36], generic chromatographic methods are often employed as a time-saving expedient [25], with the purpose of minimizing the time spent developing new analytical methods prior to each analysis session. This generic or more universal approach serves to cover a broad spectrum of compound classes, but can be also used as the starting point during method development for the analysis of other related analytes beyond the initial target
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[2].
Previously published generic methods are directed towards quantitative assays, reaction monitoring, impurity mapping and other analytical applications. However, this concept has not yet been applied to scale-up separation processes, where turnaround time, costsavings and method “greenness” are of paramount importance in the delivery of highly pure pharmaceutical drugs and synthetic intermediates. Large scale purification
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laboratories are typically a very expensive commodity, where method development and
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optimization are significantly more costly than their analytical counterparts. Separation of
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complex mixtures of closely related species can already be challenging even using stateof-the-art UHPLC on sub-2μm particle stationary phases or Coreshell column
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technologies. Handling these mixtures at preparative scale is more problematic since UHPLC columns and instrumentation are not suited for purification purposes. In addition
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to the aforementioned limitations, scale-up separations encompass additional challenges
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including sample solubility, loading optimization, fraction collection, waste accumulation and disposal; and equally important, the drying process. In this regard, developing a
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chromatographic method that combines the use of a cost-effective stationary phase (available in bulk format) and an easily removed mobile phase eluent without the need of
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a desalting requisite prior to drying, makes the difference between success or failure in the pursuit of a feasible scale up purification. Dealing with chromatographic analysis of nucleobases, sugars or compounds containing
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phosphate groups can be very challenging due to the strong polarity of these species. Separation and analysis of nucleotides (compounds containing all of the aforementioned molecular subunits) can be nightmarish to even the most skilled chromatographers. In many instances, chromatographic resolution of multicomponent nucleotide mixtures have been identified as an extremely challenging task, requiring the use of “non-conventional”
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liquid chromatography approaches in order to achieve acceptable retention, peak shape, and selectivity, e.g. hydrophilic interaction liquid chromatography (HILIC), enhancedfluidity liquid chromatography (EFLC), amongst others [37-42]. Most of the prior efforts at separating nucleotide mixtures focused on a very narrow group of nucleotides at analytical scale and do not have the potential for use in a more generic way when
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working with the diverse group nucleotides commonly found in pharmaceutical research and development. More importantly, these analytical scale separations cannot be successfully translated to large scale separations.
In this study, a novel strong anion-exchange chromatography procedure that meets all of the key requisites for the separation of over 20 of the most common nucleotides used as starting materials and synthetic intermediates in PR&D is reported. The use of this
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simple, efficient and cost-effective generic IEC method for the purification of
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multicomponent nucleotide mixtures is demonstrated with scale up data provided to
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enable its application in both analytical and large scale separation laboratories.
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2. Experimental
2.1. Instrumentation
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Anion-exchange chromatography screening and optimization experiments were
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performed on an Agilent 1260 system (Agilent Technologies, Palo Alto, CA, USA). The Agilent system was comprised of a G1311B quaternary pump, a G1329B ALS
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autosampler, a G1330B thermostat, a G4212B diode array detector, a G1316C 1260 column compartment, a G1364C 1260 FC-AS fraction collector, and a G1330B
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thermostat. Anion-exchange scale up demonstration and purification experiments were performed on both an Agilent semi-prep 1200 system (Agilent Technologies, Palo Alto, CA, USA) and an AKTA Pilot preparative system. The Agilent system was comprised of
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two G1361A prep pumps, a G2258A ALS autosampler, a G1315D diode array detector, and two G1354B Prep FC fraction collectors. The AKTA Pilot (GE Healthcare Life Sciences, Marlborough, MA, USA) was self-contained system with both UV and conductivity detection. The AKTA system was controlled by GE Healthcare Life Sciences UNICORN software, version 5.31 (GE Healthcare Life Sciences), a G1367A
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WPALS autosampler, a G1316A column compartment, and a G1315B diode array detector. 2.2 Chemicals and reagents The names and the structures of the compounds used in this study are illustrated in Figure
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1. Compounds 1-8 were obtained in house. Compounds 9-20 were obtained from SigmaAldrich, Inc. (St Louis, MO, USA). 2.3. Preparation of buffer solutions
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1 Molar (1 M) ammonium bicarbonate (NH4HCO3) in water (H2O); solution pH = 7.9: 79.06 grams (g) of NH4HCO3 were dissolved in 1 liter (L) of Millipore HPLC grade water. Solution is degassed for 20 min (vacuum, 27 torr).
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20 mM Tris Buffer in H2O solution, pH = 8: 2.42 g of Tris Base (tris-(hydroxymethyl)aminomethane, MW = 121.14 g/mole), were dissolved in 1L Millipore HPLC grade
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water.
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20 mM Tris, 0.5 M NaCl Buffer in H2O solution, pH = 8: 2.42 g of Tris Base (tris(hydroxymethyl)-aminomethane, MW = 121.14 g/mole) and 29.22 g NaCl were
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dissolved in 1L Millipore HPLC grade water.
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1 M ammonium acetate (CH3CO2NH4) in H2O solution, pH=6.88: 77 g of ammonium acetate, (MW = 77.08 g/mole) were dissolved in 1L Millipore HPLC grade water. 20 mM phosphate buffer in H2O solution, pH = 7.5: 2.31 g of Na2HPO4 (sodium
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phosphate, dibasic, (MW = 141.96 g/mole) and 0.52 g of NaH2PO4-H2O (sodium phosphate, mono basic, mono hydrate, MW = 137.99 g/mole) were dissolved in 1L
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Millipore HPLC grade water. 20 mM phosphate buffer, 1 M NaCl in H2O solution, pH = 7.5: 2.31 g of Na2HPO4 (sodium phosphate, dibasic, MW = 141.96 g/mole), 0.52 g of
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NaH2PO4-H2O (sodium phosphate, mono basic, mono hydrate, MW = 137.99 g/mole), and 58.44 g of NaCl were dissolved in 1L Millipore HPLC grade water. 20 mM phosphate buffer in water (pH 8.5) solution: 2.78 g of Na2HPO4 dibasic (MW 141.96 g/mole) and 0.06 g of NaH2PO4-H2O mono (MW137.99 g/mole) were dissolved in 1L Millipore water. 20 mM phosphate buffer in water (pH 8.5) solution with 1 M
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NaCl: 2.78 g of Na2HPO4 dibasic (MW 141.96 g/mole), 0.06 g of NaH2PO4-H2O mono (MW137.99 g/mole), and 58.44 g of NaCl were dissolved in 1L Millipore water. 2.4. Screening for stationary phases and mobile phases Chromatographic column screening was performed to determine optimal
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chromatographic conditions for preparative ion-exchange chromatography (IEC).
Initially, two anion-exchange (AEX) columns were screened in conjunction with three
separate mobile phase combinations. The AEX columns selected for the screening were the Proteomix SAX-NP10 (4.6 x 50 mm) and the WAX-10NP (4.6 x 50 mm), both
purchased from Sepax. The first mobile phase examined consisted of eluents (A) 20 mM Tris buffer (pH 8.0) and (B) 20 mM Tris, 0.5 M NaCl buffer, (pH 8.0). A gradient
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elution profile (30 minutes) was executed as follows: 0 min (0% B), 20 min (100% B),
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20.1-30 min (0% B). The second mobile phase examined consisted of eluents (A) water and (B) 1 M ammonium acetate in water (pH 6.7) and the third mobile phase examined
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was (A) water and (B) 1M ammonium bicarbonate in water (pH 8.01). Gradient elution
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profiles (45 minutes) for the second and third mobile phases were executed as follows: 02 min (1% B), 2-22 min (100% B), 22.5-45 min (1% B). All screens consisted of 10 µL
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injections performed in duplicate with a flow rate of 0.5 mL/min, column temperature of
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25 °C, and UV detection at 260 nm (reference signal at 360 nm). The next round of column screening was carried out with the following strong anionexchange (SAX) columns; the UniCore 10Q, AEX column (4.6 x 150 mm, 10 µm
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particles) purchased from Nexgen and the TSKgel SuperQ 5PW (10) (4.6 x 150 mm, 10 µm particles) purchased from TOSOH. As before, three mobile phase combinations were
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applied with these columns. The eluents employed for the screening were as follows: Eluent 1 - (A) 20 mM phosphate buffer (pH 8.5), (B) 20 mM phosphate buffer (pH 8.5),
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1 M NaCl; Eluent 2 – (A) Water, (B) 1 M ammonium bicarbonate (pH 8.0); Eluent 3 – (A) 20 mM phosphate buffer (pH 7.5), (B) 20 mM phosphate buffer (pH 7.5), 1 M NaCl. The gradient elution profiles for all three mobile phases were executed as follows: 0 min (5% B), 80-100 min (100% B), 100.1-120 min (5% B). All of these column screens were conducted at a flow rate of 0.27 mL/min, column and sample temperatures of 22°C and 45°C, respectively, and UV detection set to 210, 254, and 280 nm. 6
2.5. Experimental conditions used for LC-Simulator modeling Upon review and evaluation of the afore-listed screening study, the following column and chromatographic conditions were selected for continued IEC method development for separation of 20 nucleotides (Fig. 1) including chromatographic modeling and simulation
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(see some chromatographic profiles in Fig. 2a): IEC resin: TOSOH TSKgel SuperQ 5PW (4.6 mm x 150 mm, 10 µm particles) column by a gradient elution using eluent A: water
and eluent B: 1 M ammonium bicarbonate (pH 8.0). Flow rate: 0.27 mL/min. Two eluent gradients, 10-90% B in 40 min and 10-90% B in 80 min, each followed by 20 min re-
equilibration, were executed at three temperatures (30, 40, and 60 °C) in order to build a 3D resolution map. The resultant experimental data was input and processed using
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ACD/LC Simulator 2015 Release (Version L10R41), Advanced Chemistry Development,
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Inc. (ACD), Toronto, Ontario, Canada, further referred to as LC Simulator.
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2.6. Optimized Anion-Exchange Chromatography method
M
The 3D resolution map was built based on the conditions listed-above, which allowed us to develop the following optimized method for separation of all 20 nucleotides using the same IEC resin and mobile phase. Gradient: 10% to 90% B in 60 min, with 20 min re-
D
equilibration time at 10% B. Flow rate: 0.27 mL/min. Column temperature: 30°C.
TE
Injection volume: 5 μL. UV detection: 254 nm. Experimental chromatogram is shown in Fig. 2b.
EP
2.7. Chromatographic conditions for isolation and purification of synthetic intermediates
CC
and process related impurities IEC scale-up purification was implemented on a 70 mm × 500 mm (20 μm particles), TOSOH TSKgel SuperQ 5PW (20) column via gradient elution chromatography (AKTA
A
Pilot) derived from the best conditions introduced above. Purification of intermediate #3: a gradient elution profile (at flow rate of 80 mL/min) was performed in which the eluent delivered was 0-32 min (30% B), 60-100 min (40% B), 100.1-120 min (100% B), 120.1150 min (30% B). Mobile phase component (A) was HPLC grade water and mobile phase component (B) was 1 M ammonium bicarbonate (pH 8.0). Sample load volume was 250 mL (4g mixture of active 25 wt% intermediate #3 in water) and the IEC column 7
and sample feed were maintained at ambient (22°C) temperature, respectively. Purification of intermediate #7: a gradient elution profile (at flow rate of 80 mL/min) was performed in which the aforementioned mobile phase delivered was 0-35 min (35% B), 60-100 min (45% B), 100.1-125 min (100% B), 125.1-155 min (35% B). In likewise fashion, the sample load volume was 250 mL (3.7 g mixture of active 27 wt%
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intermediate #7 in water) under ambient (22°C) column and sample feed temperatures, respectively.
A smaller scale IEC purification was carried out on a 35 mm × 250 mm (20 μm particles), TOSOH TSKgel SuperQ 5PW (20 μm) column via gradient elution
chromatography (Agilent 1200 series) under the aforementioned method conditions.
U
Purification of process related impurity #4: a gradient elution profile (at flow rate of 20 mL/min) was performed in which the mobile phase (mobile phase (A) was water and
N
mobile phase (B) was 1 M ammonium bicarbonate (pH 8.0)) delivered was 0-16 min
A
(30% B), 40-60 min (40% B), 60.1-70 min (100% B). The sample load volume was 20
M
mL (50 mg of desired material #4 in water) with an ambient temperature of 22°C for the column and sample feed, respectively. Purification of process related impurity #8: a
D
gradient elution profile (at flow rate of 20 mL/min) was performed in which the mobile phase (mobile phase (A) was water and mobile phase (B) was 1 M ammonium
TE
bicarbonate (pH 8.0) delivered was 0-42 min (35% B), 60 min (42% B), 60.1-70 min (100% B). The sample load volume was 14 mL (40 mg of desired material #8 in water)
EP
with an ambient temperature of 22 °C for the column and sample feed, respectively.
CC
3. Results and Discussion Separation and analysis of multicomponent mixtures of nucleotides is never easy, and requires chromatographic approaches beyond conventional reversed phase liquid
A
chromatography (RPLC). Other well-established techniques in the pharmaceutical industry such as normal phase liquid chromatography (NPLC) and supercritical fluid chromatography (SFC) are often inapplicable due to exceedingly strong retention, aggravated tailing effects, and poor chromatographic performance. The development and optimization of a generic chromatographic method for the separation and purification of multiple nucleotides in a single experimental run is essential for a fast and reliable 8
turnaround of analytical results and batch purification deliveries. All of the 20 nucleotides selected in this investigation for method development form part of a comprehensive list of starting materials and intermediates that have been used by synthetic and process chemists in our laboratories for pharmaceutical research and development. The list also includes dinucleotides and other structurally diverse
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nucleotides containing different nucleobases and number of phosphate groups (Figure 1). We examined each of these mixtures using several chromatographic screening systems that are routinely used within these laboratories for the development of analytical
methods[4,43,44]. As expected, RPLC and SFC showed very poor chromatographic
performance and inadequate resolution. IEC screening, by contrast, provided conditions for the separation of multiple nucleotides shown in figure 1. A complete listing of the
U
columns, eluents, and methods used in the screening experiments is described in the
N
Experimental Section. It is important to point out that selection of the best screening hits
A
for method development is based on criteria that enable both analytical and large scale separations, e.g. peak shape, retention, separation, resin cost and availability in bulk
M
format; and equally important, a mobile phase that can be directly removed by conventional drying technologies. In this regard, TOSOH TSKgel SuperQ-5PW resin
D
proved to be an excellent stationary phase candidate for method development and
TE
optimization. These columns are packed with porous hydrophilic polymer (polymethacrylate based) beads which are modified with a strong anion-exchange group
EP
(trimethyl-amino ligands). TOSOH TSKgel SuperQ-5PW resin shows excellent chromatographic performance for the separation of all 20 nucleotides when combined
CC
with a fully aqueous ammonium bicarbonate-based eluent. The use of organic eluents in the mobile phase is not required which serves to reduce cost and environmental impact,
A
something of vital importance in the implementation of large scale purification processes. The aforementioned screening results were subsequently optimized in order to develop a method that separates all of the 20 nucleotides in a single experimental run. Such generic chromatographic conditions could be effectively applied for the isolation and purification of nucleotide species beyond the scope of this list, where reduction in method development efforts can lead to significant time savings when multiplied across the many 9
samples submitted for purification. The results outlined in Figure 2a illustrate initial attempts at the separation of the entire mixture. Compounds were subdivided into five different mixtures to minimize sample degradation and simplify peak identification. The resultant experimental data was input and processed using ACD/LC Simulator software. Two eluent gradients: 10-90%B in 40 min and 10-90%B in 80 min, each followed by 20
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min re-equilibration, were run at three different temperatures (30, 40, and 60 °C) in order to construct a 3D resolution map that was subsequently used for the prediction of chromatographic conditions and elution profiles depicted in figure 2b.
The 3D resolution map shows a very small and narrow area (orange) of robust conditions allowing for the efficient and convenient resolution of all 20 nucleotides using a linear
U
gradient elution, in particular, all of the critical pairs that are very difficult to separate (e.g. nucleotides 2 and 5, 3 and 6) using the initial gradient and column temperature.
N
Figure 2b also shows the overlaid chromatographic profiles from both simulated and
A
experimental data illustrating the power of modern chromatography simulation and
M
modeling software when applied for method development. Interestingly, some critical pair separations exhibited elution order variations in going from initial screening to the
D
optimized conditions. As is expected for IEC, all of the dinucleotides are more retained, with increased overall retention in concert with the number of phosphate groups. These
TE
simulated chromatographic data were compared to experimental outcomes. The comparisons outlined in table 1 show an excellent match, with overall ∆tR differences
EP
below 0.26 min between predicted and experimental data. Following suit, the aforementioned analytical IEC conditions (resin and buffer eluent)
CC
were effectively used as the starting point during method development for the isolation and purification of nucleotide intermediates and process related impurities. This approach
A
serves to streamline scale up transition and speed up purification turnarounds, which leads to significant time savings when multiplied across many pipeline programs. Figure 3 shows an impressive collection of highly efficient separations across a challenging set of closely related nucleotides in reaction mixtures. All of the optimizations were performed by minimal modification of the mobile phase gradient, column dimensions and flow rate while retaining the same IEC resin and ammonium bicarbonate based eluent. 10
A baseline separation of intermediate #3 from a very complex reaction mixture (Fig. 3a) was achieved on large scale using readily available preparative FPLC instrumentation (AKTA Pilot) on a 70 mm I.D. x 500 mm length (20 μm) column. This method allowed us to process 4g of crude per injection enabling the delivery of 12g of intermediate #3 (96% purity) from a very complex reaction mixture. It is important to point out that the
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same method was applied to the isolation of AMP and ADP (nucleotide #1 and #2) which are closely related process byproducts formed in the reaction. Figure 3b illustrates
another successful scale up purification easily accomplished by minor modification of gradient conditions using the same column and buffer. This method was effectively
implemented to obtain 34g of intermediate #7 with 99% purity, an excellent result from another complex mixture requiring no further purification. Interestingly, this
U
chromatographic procedure is also very effective for the separation of other nucleotides
N
formed in the same reaction (e.g. compounds #5 and #6). In another example depicted in figure 3c, a similar approach using readily available semipreparative HPLC
A
instrumentation ((Agilent 1200 series) on a 35 mm I.D. x 250 mm length (20 μm)
M
column) was applied to the isolation of a process related impurity for use as reference standard (compound # 4: 60 mg isolated with 98% purity). Note that the same IEC
D
conditions can be used for separation of compound # 3 which is also a reaction product.
TE
The final example shown in figure 3d is related to the isolation of another impurity (compound #8) for reference standard purposes. The same column with a modified step gradient was utilized to deliver 50 mg of this impurity (99% purity). It is important to
EP
highlight that all of these rich cut fractions contain the desired analytes in a fully aqueous ammonium bicarbonate buffer and can be directly dried by lyophilization without the
CC
need for a desalting procedure after purification. This is a critical feature that makes these large scale separation methods so effective in accelerating purification deliveries and
A
minimizing chemical degradation. In addition, the use of organic solvents such as acetonitrile and alcohols are not required which serves to reduce toxic waste and minimize environmental impact.
4. Conclusions 11
Pharmaceutical, agricultural, biomedical, and other research and development disciplines, often involve the extensive use of nucleotides as building blocks for synthetic route development. Consequently, chemists have to spend a considerable amount of time and resources developing analytical and preparative chromatographic methods prior to each purification session. To better meet these needs, a simple, efficient, and cost-effective
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IEC method for separation and purification of over 20 nucleotides was developed and implemented. Our method combines the use of a Tosoh TSKgel SuperQ-5PW in
conjunction with a fully aqueous eluent profile (ammonium bicarbonate-based) that
enables a straightforward scale up transition and convenient drying process with minimal environmental impact. This generic method was optimized using ACD Labs
chromatography simulation software and successfully applied to the large scale
U
purification of complex mixtures of nucleotides by using readily available preparative
N
FPLC instrumentation. In addition, these IEC conditions combined with ACD Labs modeling can be effectively used as a starting point during method development for the
M
A
isolation of other highly polar nucleotide species beyond the list described in this study. Acknowledgements
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We thank Dr. Mirlinda Biba (Merck & Co, Inc.) and collaborators at Sepax Technologies for fruitful discussions.
12
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Fig. 1. Structures and names of nucleotides used in this study
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Fig. 2. Method development for separation of 20 nucleotides from modeling (a) to experimental conditions (b). All chromatographic conditions applied to build the modeling and 3D resolution map are described in the Experimental section, as well as the final optimized conditions.
A
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A
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Fig. 3. Purification of nucleotides intermediates and impurities used/formed during the synthesis of more complex molecules at MSD Research Laboratories. IEC resin: TSKgel SuperQ-5PW (20 μm), eluent A: H2O, Eluent B: 1 M ammonium bicarbonate, temperature: ambient, detection: 254 nm. Injection volume and Gradient elution as follow: a) 70 mm I.D. x 500 mm length column; 16 mg/mL, 250 mL injected; 30 to 40 % eluent B in 100 min. b) 70 x 500 mm column; 14.8 mg/mL, 250 mL injected; 35 to 45 % eluent B in 100 min. c) 35 x 250 mm column; 5 mg/mL, 20 mL injected; 30 to 40 % eluent B in 60 min. d) 35 x 250 mm column; 4.0 mg/mL, 14 mL injected; 35 to 42 % eluent B in 60 min, then go to 100% B in 0.1 min, with a final 10 min hold. Chromatographic conditions are outlined in more detail in the Experimental section.
17
Fig. 1.
Peak #
Compound name 2‘R-adenosine-5-(1-R-momonophosphate)
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1 2
2‘R-adenosine-5-(1-R-diphosphate)
3
2‘R-adenosine-5-(1-R-triphosphate)
4
2‘R-adenosine-5-(1-R-tetraphosphate)
5
3‘R-guanosine-5'-R-monophosphate
6
3‘R-guanosine-5'-R-diphosphate
7
3‘R-guanosine-5'-R-triphosphate
8
3‘R-guanosine-5'-R-tetraphosphate
9
Adenosine 3′,5′-cyclic monophosphate sodium salt
10
Adenosine 5′-monophosphate sodium
11
Adenosine 5′-diphosphate sodium
12
Adenosine 5′-triphosphate disodium salt
13
15
Guanosine 5′-diphosphate sodium salt
16
Guanosine 5′-triphosphate sodium salt hydrate
17 18
β-Nicotinamide adenine dinucleotide sodium salt β-Nicotinamide adenine dinucleotide phosphate sodium salt hydrate
19
Nicotinamide guanine dinucleotide sodium salt
20
Nicotinamide hypoxanthine dinucleotide sodium salt
A
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A
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14
Guanosine 3′,5′-cyclic monophosphate sodium salt Guanosine 5′-monophosphate disodium salt hydrate
18
Fig. 2.
2a) Structure of stationary phase and experiments to build the model 12
10 11
1500
15
16
14
experimental
1000
Strong anion exchanger phase (note: R’=propriety)
17
19 20
0 5
15
70
0.00010
17 17
16 16 44 33
0.00006
19 19
22
14 14
0.00005
88 77
66 55
11
99
0.00004
55
65
1.50
55
1.43 1.36 1.29 1.21 1.14 1.07 1.00
50
0.93 0.86
45
0.79 0.71
A
13
45
U
15 15
Column Temperature, °C
20 20
0.00007
35
1.57
60
12 12
10 10
Mix 5
N
11 11
Mix 4 Mix 2 Mix 3 Mix 1
1.64
65
18 18
0.00008
8
2
1.72
Predicted Experimental
0.00009
7
18
25
2b) Optimum conditions
3 6
5
1
500
4
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13 9
0.00003
0.64
40
0.00002
0.57 0.50 0.43
35
15
20
25
30
35
40
45
50
55
60 min
0.36 0.29 0.21
30
0.14 0.07 8
16
24
32
40
48
56
64
72
80
88
96
104
112
120 0.00
Time, min
A
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10
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0.00001
19
Fig. 3.
Resin: TSKgel SuperQ-5PW
Mobile phase: NH4HCO3 based eluent
a) Large scale purification of Merck intermediate #3
Crude: 47.5 LCAP, 25 wt% Crude: 48.5g, purified: 12.2g Final Purity: 96 LCAP
3000
Desired intermediate #3
#2
2000
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#1
1000
0 0
50
100
150
b) Large scale purification of Merck intermediate #7 Desired intermediate #7
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Crude: 75 LCAP, 27 wt% Crude: 123g, purified: 34.2g Final Purity: 99 LCAP
3000
N
#5 2000
M
A
#6
1000
0 0
50
100
150
c) Purification of Merck impurity #4 for reference standard
D
1200 Crude: 51 LCAP Purified: 60mg Final Purity: 98 LCAP
Desired imp #4
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800
#3
0
10
20
30
40
CC
0
EP
400
50
60
d) Purification of Merck impurity #8 for reference standard Crude: 75 LCAP Purified: 50mg Final Purity: 99 LCAP
Desired imp #8
A
mAU
1000
#7 500
0 0
20
40
60
min
20
Table 1. Summary of predicted and experimental retention time values for all 20 nucleotides
tR, predicted
tR,experimental ∆tR (%)
1 2 3 4 5 6 7 8 9 10 11 12 13
43.84 50.17 53.57 55.86 50.50 53.80 57.06 58.67 25.56 28.81 37.98 42.99 35.14
43.63 49.94 53.34 55.60 50.27 53.57 56.83 58.42 25.50 28.83 37.92 42.88 34.98
0.48 0.46 0.43 0.47 0.46 0.43 0.40 0.43 0.24 0.07 0.16 0.26 0.75
14 15 16 17 18 19 20
35.73 42.73 46.84 10.61 15.73 17.99 31.97
36.00 42.53 46.61 10.60 15.70 18.01 31.86
0.46 0.47 0.49 0.09 0.35 0.19 0.11
A
CC
EP
TE
D
M
A
N
U
SC RI PT
Peak #
21