Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 316–324

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Supercritical fluid chromatography for GMP analysis in support of pharmaceutical development and manufacturing activities Michael B. Hicks ∗ , Erik L. Regalado, Feng Tan, Xiaoyi Gong, Christopher J. Welch Department of Process & Analytical Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA

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Article history: Received 14 July 2015 Received in revised form 9 September 2015 Accepted 13 September 2015 Available online 15 September 2015 Keywords: Supercritical fluid chromatography GMP SFC Enantiomers Pharmaceutical impurities Chiral stationary phases Method validation

a b s t r a c t Supercritical fluid chromatography (SFC) has long been a preferred method for enantiopurity analysis in support of pharmaceutical discovery and development, but implementation of the technique in regulated GMP laboratories has been somewhat slow, owing to limitations in instrument sensitivity, reproducibility, accuracy and robustness. In recent years, commercialization of next generation analytical SFC instrumentation has addressed previous shortcomings, making the technique better suited for GMP analysis. In this study we investigate the use of modern SFC for enantiopurity analysis of several pharmaceutical intermediates and compare the results with the conventional HPLC approaches historically used for analysis in a GMP setting. The findings clearly illustrate that modern SFC now exhibits improved precision, reproducibility, accuracy and robustness; also providing superior resolution and peak capacity compared to HPLC. Based on these findings, the use of modern chiral SFC is recommended for GMP studies of stereochemistry in pharmaceutical development and manufacturing. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Over the past two decades supercritical fluid chromatography (SFC) has emerged as the universally preferred analytical technique for assaying stereoisomeric mixtures, largely displacing previously used normal and reversed phase chiral chromatographic methods [1–3]. Today, most laboratories engaged in the analysis of enantiopurity to support discovery and development of pharmaceuticals and fine chemicals use SFC with chiral stationary phases (CSPs) as the method of choice [4–6]. Ongoing improvements in analytical SFC instrumentation (pumps, autosamplers, mixers, backpressure regulators, minimized extra column volume, etc.) [7–10] combined with improvements in column and stationary phase technologies (smaller particles, porous shell technologies, new chiral stationary phases, etc.) [11–13] have enabled high performance and high speed enantioseparations [14] that compare very favorably with traditional liquid chromatographic approaches [15–17]. The first generation of SFC instrumentation suffered from drawbacks such as poor injection reproducibility, excessive detection noise due to back pressure regulator variation, inefficient pump damping, and overall poor detection sensitivity, making SFC less

∗ Corresponding author. E-mail address: [email protected] (M.B. Hicks). http://dx.doi.org/10.1016/j.jpba.2015.09.014 0731-7085/© 2015 Elsevier B.V. All rights reserved.

attractive in analytical laboratories regulated by Good Manufacturing Practice (GMP) where method precision and sensitivity for low level chiral impurities is routinely required for supporting manufacturing operations. Customized instrumentation allowed many of these shortcomings to be overcome [18,19] but required expert solutions. Consequently, many laboratories carrying out GMP testing of stereoisomeric mixtures currently use traditional normal phase or reversed phase chiral chromatographic methods. These methods often require the conversion of the fast and efficient SFC assay used to support discovery and development into a more sensitive and reproducible (but often slower and less efficient) HPLC assay. Recent commercialization of next generation analytical SFC instrumentation, e.g. the Aurora-Agilent and the Acquity UPC2 instruments, [9,20] now provides ready access to higher sensitivity, robustness and reproducibility, potentially allowing SFC to be routinely used in GMP analytical support for manufacturing activities [21–23]. In this study we evaluate the suitability of two modern analytical SFC instruments: the Agilent1260 Infinity Analytical SFC System (formerly Aurora Fusion A5) and the Acquity Ultra Performance Convergence Chromatography System (UPC2 ) for performing routine GMP assays in the modern drug development and manufacturing environment.

M.B. Hicks et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 316–324

2. Experimental 2.1. Instrumentation Experiments were carried out using the available modern SFC systems in our labs: (1) Agilent-Aurora A5 Fusion SFC module Agilent 1100HPLC (Agilent Technologies, Palo Alto, CA, USA) equipped with a vacuum degasser, column temperature compartment and diode array detector. This system was controlled by Agilent Chem Station B.03.01, SR1 using Open Labs and connected through eSatin data converter to Empower GMP compliant software. Typical modifications to the standing 1100 system includes replacing standard green tubing (0.17 ID) with shorter minimum diameters (0.05 ID or 0.07 ID) tubing to the column compartment to the detector and replacing the diode array detector standard low pressure, 3 mm flow cell with the SFC recommended high pressure, larger 10 mm width flow cell. These modifications have become the standard features in recent Agilent 1260 SFC Control modules series, with an insulated high-pressure flow cell already included. (2). Acquity Ultra-Performance Convergence Chromatography System (UPC2 ), (Waters Corp., Milford, MA, USA) system equipped with a fluid delivery module (a liquid CO2 pump and a modifier pump), a sampler manager (FL autosampler), eight column thermostat compartment including heat-insulated pre-column tubing, a photodiode array detector, Empower GMP compliant Software all fully firmware controlled with the Waters the convergence manager to optimize all pressure, temperature and flow parameters. This Acquity UPC2 chiral SFC includes a screening platform with eight column capacity: two channels without columns, for achiral columns inter-changing flexibility and six 4.6 × 150 mm, 3 ␮m columns operated at 3 mL/min (6 columns running serially at 6 min/column for the method screen at total of 36 min). 2.2. Chemicals and reagents Methanol, ethanol, isopropanol, acetonitrile (HPLC Grade) were purchased from Thermo Fisher Scientific (Fair Lawn, NJ, USA). Merck research compounds MR-1 to MR-5 were all obtained from the Merck Compound Resources. Industrial grade CO2 was obtained from Air Gas (Radnor, PA). Additives to the SFC modifiers were triethylamine (TEA) from Thermo Fisher Scientific and isobutyl amine from Sigma–Aldrich (St. Louis, MO, USA). 2.3. Sample solutions Solution concentrations for HPLC were between 0.2 and 0.5 mg/mL prepared with 20 mg–50 mg of API solid to 100 mL of diluent. The diluent was 50% acetonitrile:50% water for all but MR-3, that used acetonitrile with 0.02 v/v% perchloric acid and MR5, that used methanol. The SFC methods used between 1.2 and 1.0 mg/mL prepared with 50 mg of solid to 50 mL of diluent. Similarly, the diluent was 50% acetonitrile:50% water for all but MR-3, that used acetonitrile with 0.02 v/v% perchloric acid and MR-5, that used methanol. Dilutions were, for example 1.0 mL in 100 mL for a 1% of stock concentration, then separately prepared volumetric dilutions were made from down to 2000× dilution for both SFC and HPLC, 0.05 v/v% with 0.5 mL of the 1.0% reference standard to 10 mL of diluent. Typical linearlity ranges covered 0.05–20% of the analyte target concentration, unless indicated. 2.4. HPLC conditions Chiral HPLC conditions are indicated per compound with normal phase or reverse phase conditions as follows: Research compound MR-1 uses an immobilized celullose, tris-(3,5-dimethylphenyl carbamate) coated selector, Chiralpak-IA (150 × 4.6 mm, 5 ␮m) at

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40 ◦ C column temperature by UV detection 277 nm, and a gradient elution from 100% A (hexane with 0.05 v/v% triethylamine (TEA)) to 60%:40% A:B where B is ethanol-0.05 v/v% TEA. MR-2 uses a cellulose,tris-(3,5-dimethylphenyl carbamate) coated selector, the Chiralcel OD-3R (150 × 4.6 mm 3 ␮m) at 30 ◦ C column temperature by UV detection 220 nm and isocratic LC conditions 44% 150 mM potassium hexafluorophosphate pH 2.1 and 56% acetonitrile. MR-3 uses a Chiralpak IA (250 × 4.6 mm, 5 ␮m) at 45 ◦ C column temperature by UV detection 254 nm with isocratic LC conditions at 20% isopropanol (0.1% TFA v/v), 80% hexane. Research compound MR4 uses a Chiralcel OD-RH (150 × 4.6 mm, 5 ␮m) at 25 ◦ C column temperature by UV detection 220 nm and a reverse phase gradient from 20% to 60% B (acetonitrile) over 30 min where A is 0.02% v/v HCLO4 + 150 mM NaClO4 in water, 80/20 (v/v). The final commercial compound MR-5 method uses a cellulose tris(3,5-dimethylphenyl carbamate) the Lux Cellulose-1 (250 × 4.6 mm, 3 ␮m) at 35 ◦ C column temperature by UV detection 350 nm with isocratic conditions at 80% 1:1 methanol:ethanol with 0.2% diethylamine, 20% heptane with 0.2% diethylamine. All HPLC methods used a 1.0 mL/min flow rate. 2.5. SFC conditions Chiral SFC conditions are indicated per compound with SFC conditions as follows: research compound MR-1 used a Chiralcel OD-H (250 × 4.6 mm 5 ␮m) at 45 ◦ C column temperature by UV detection at 277 nm with isocratic conditions at 75% CO2 (200 bar) and 25% methanol containing 25 mM isobutyl amine. MR-2 uses a Chiralcel OD-3 (150 × 4.6 mm 3 ␮m) at 27 ◦ C column temperature by UV detection at 254 nm and isocratic conditions at 70% CO2 (150 bar) and 30% methanol. MR-3 uses a an amylose, tris-(3,5-dimethyl carbamate) coated selector Chiralpak AD-3, (100 × 4.6 mm, 3 ␮m) at 35 ◦ C column temperature by UV detection 210 nm and gradient conditions at 2.5 mL/min 10% methanol containing 25 mM isobutyl amine for 3 min then to 40% at 5%/min (200 bar). Research compound MR-4 uses a Chiralpak AD-3 (150 × 4.6 mm, 3 ␮m) at 40 ◦ C column temperature by UV detection 220 nm and isocratic conditions at 65% CO2 (220 bar) and 35% isopropanol. MR-5 uses a Regis Technology, RegisCell (250 × 4.6 mm, 5 ␮m) a tris-(3,5dimethylphenyl) carbamoyl polysaccaride coated high-purity silica column at 35 ◦ C and 350 nm UV detection and isocratic conditions at 65% CO2 (200 bar) and 35% methanol containing 25 mM isobutylamine. All SFC methods used a 3.0 mL/min flow rate, except where indicated. All conditions here that refer to “supercritical” as a convention are actually using “subcritical” conditions below 40 ◦ C and ∼70% CO2 . 3. Results and discussion 3.1. Sensitivity, linearity and range of quantitation using the Agilent-Aurora-Fusion A5 The Aurora-Agilent Fusion A5 is a modular SFC system introduced in 2009, which incorporates a free-standing CO2 module that allows existing HPLC instruments to be converted for SFC use. For older HPLC instruments, simple modifications of connecting tubing, flow cells and check-valves have been shown to afford ready access to improved performance (see Section 2). Fig. 1a shows an SFC determination of the enantiomeric purity of a development compound, MR-1, highlighting the low peak to peak noise level (∼0.02 mAU) relative to the peak height of 0.05% of the minor enantiomer (∼0.5 mAU). The resulting signal to noise ratio (s/n) of 25 easily exceeds the limit of S/N ≥ 10 that is required for appropriate quantitation. This sensitivity allows reliable measurement of enantiopurity ranges that are critical for GMP pharmaceuti-

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Fig. 1. Chromatographic separation of the enantiomers by SFC for ∼1 mg/mL of a pharmaceutical development compound MR-1 by SFC and HPLC. (a) Low baseline noise and excellent signal to noise allow low level quantitation of minor enantiomer. Chiral impurity profiling over 2 min with a target API signal of 1AU for the major component (Merck research and development compound, MR-1 by chiral SFC using the Agilent Baseline Noise Test looking at the highest and lowest signal across a representative time range in the method from 8.0 to 10 min. (b) Comparison of chiral SFC and HPLC assays for enantiopurity analysis of MR-1. Chromatographic conditions for both methods are described in the experimental section. (c) General overview of the HPLC and SFC chiral purity method sensitivity and linearity for MR-1. [Note the molar extinction coefficient for this compound in methanol is 5× greater than in the 25%:75% methanol:CO2 mixture resulting in a greater target mg/mL for SFC].

cal analysis (typically up to 99.9% ee). The actual signal to noise ratios obtained for different analytes will of course be dependent on a variety of factors relating to chromatographic separation, detection response factors, etc., however, in our experience, SFC assays using this instrument are often suitable for enantiopurity determination of pharmaceuticals in regulated GMP laborato-

ries, now having equivalent sensitivity to reverse phase chiral HPLC. By comparison, normal phase HPLC results for this API (Fig. 1b and c) show that while HPLC provides a similar limit of quantitation at 0.4 ␮g/mL, the inability to fully separate the enantiomers (significant peak tailing and poor resolution) results in poor chro-

M.B. Hicks et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 316–324 Table 1a Analytical performance for spiked recovery of MR-1. Spiked impurity (%)

SFC recovery (%)

0.10 0.15 0.24

3.3. Sensitivity linearity and range of quantitation using the Acquity UPC2 Criteria recovery (%) ≤85%

97.8 107.1 101.8

Table 1b Inter-analyst assay repeatability for MR-1. Analyst Minor (a RSD%)

Criteria (Minor, RSD%)

1 2

≤25%

3.83 5.31

Major (a RSD%) 0.04 0.04

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Criteria (Major, RSD%) ≤0.5%

a Experiments were performed using nine separate sample preparations per analyst (n = 9).

matographic performance that subsequently limits the required upper linear range for the major enantiomer from 0.4 to 10 ␮g/mL in order to maintain the required resolution of the minor impurity. In comparison, the SFC assay provides improved resolution of the enantiomer and several minor impurities along with a shorter run time and a much greater linear range, affording an overall superior ability to detect and quantitate both enantiomers with good method response precision (≤25% () ≤10%). In addition, the higher chromatographic efficiency (theoretical plates, N) and the superior resolution of the SFC method allowed the appropriate upper linear range, to 120% of target concentration or 1200 ␮g/mL (Fig. 1c). These combined advantages led to the selection of SFC as the preferred method for the registration phase enantiopurity analysis for MR-1. 3.2. Specificity/accuracy/precision/repeatability using the Agilent-Aurora-Fusion A5 In addition to good detection sensitivity and improved linearity through better impurity resolution, accuracy and specificity quantitation is critically important for quantitative chiral GMP analysis. The accuracy of a GMP method is commonly investigated using spike and recovery experiments, where known amounts of various components and impurities are spiked into a sample which is then analyzed to determine recovery. The spike and recovery results carried out for the MR-1 enantiopurity analysis at the required concentrations have acceptable recoveries well within ±15% of the expected recovery (Table 1a). The method repeatability is another important criterion for validation of GMP analytical methods, with consistent retention time and peak area between injections and over prolonged periods of time; an important parameter to ensure correct identification of chromatographic peaks and accurate assay results. Table 1b demonstrates the repeatability of the assay and how the method performs between two analysts with the criteria for peak area percent determinations for both the API and the minor enantiomer being met for nine separate preparations of MR1 spiked at 0.1% (1 ␮g/mL) of the target concentration. In addition to the repeatability, the inter-analyst precision of the method was also found to be excellent (Table 1c) for appropriate inter-analyst precision. Similarly, a series of ten test injections each carried out on the Agilent/Aurora SFC system showed less than 0.32% RSD variation in peak area and less than 0.77% RSD variation in retention time for isocratic runs and less than 0.48% RSD variation in peak area and less than 0.65% RSD variation in retention time for gradient runs. Together, these results clearly show that given appropriate conditions; modular SFC systems like the Fusion A5 can appropriately provide separations methods suitable for GMP analysis.

The recently introduced Aquity UPC2 is the first commercial SFC instrument to incorporate modern ‘post UPLC’ design principles such as radical minimization of mixing and extra-column volumes and incorporation of an improved modifier pump and PDA detector [20]. In this instrument, the liquid CO2 handlers are chilled and insulated for very precise density control of subcritical CO2 entering the pump to allow accurate fine tuning of modifier ratios as low as 2%, a substantial improvement over previously available systems. A separate auxiliary injection valve in the Waters dual injector minimizes injection pulsation and mitigates carryover while the primary valve is vented to waste under atmospheric pressure. This allows for partial loop SFC injections, an important feature that affords increased flexibility. The PDA preserves the 10 mm path length of previous instruments but offers a low volume capillary stainless steel flow cell for low dispersion and rapid thermal equilibration. A comparison of the enantioseparation of the drug, MR-2 using either SFC or conventional HPLC is illustrated in Fig. 2. The SFC method can easily detect

Supercritical fluid chromatography for GMP analysis in support of pharmaceutical development and manufacturing activities.

Supercritical fluid chromatography (SFC) has long been a preferred method for enantiopurity analysis in support of pharmaceutical discovery and develo...
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