ABSTRACT

The Ames Salmonella/microsome mutagenicity assay (Ames test), a short-term bacterial reverse mutation assay, is a type of genotoxicity assay used worldwide and recommended by the Food and Drug Administration and International Conference on Harmonization for an investigational new drug application [1-5] In addition to an Ames test conducted according to good laboratory practice (GLP) procedures, an abbreviated or “screening” version of the Ames test is typically used in lead optimization studies to help determine which molecule to bring forward in development Currently, no industry-wide guidelines specifically define the purity requirement of compounds for this assay High-purity (both chiral and achiral) materials are recommended in lead optimization studies by many pharmaceutical companies to eliminate false positive results The challenge in purification lies in removing not only the chiral impurities but also an array of achiral impurities, which are not well characterized at this point

Since the introduction of a commercially available instrument in the early 1990s, supercritical fluid chromatography (SFC) has been gradually accepted as an efficient tool for purification SFC is a type of normal phase chromatography utilizing compressed carbon dioxide in conjunction with organic solvent as the mobile phase High diffusivity and low viscosity of the mobile phase allow for rapid flow rates, leading to reduced analysis time without sacrificing efficiency [6-9] SFC is suitable for purifying materials with stringent purity criteria and aggressive time lines Advantages include rapid method development and relatively simple scale-up The reduced amount of solvent waste generated by chromatographic separation and the smaller volume of purified fractions are also outstanding features of this technique Additionally, volatile organic solvents such as methanol (MeOH), ethanol (EtOH), or isopropanol (IPOH) utilized as organic additives to carbon dioxide are easier to remove compared to an aqueous mobile phase [10-12] The use of volatile base additives, such as diethylamine (DEA) or isopropylamine, in the SFC mobile phase can dramatically enhance the peak shape of basic compounds, resulting in improved resolution

Purity of lead compounds is better assessed using at least two orthogonal systems such as reversed phase liquid chromatography-mass spectrometry (LC/MS) and supercritical fluid chromatography-mass spectrometry (SFC/MS) systems In our laboratory, SFC/MS is the primary means for the assessment of chiral purity as well as the tool to provide additional information on achiral purity Achiral purity is generally assessed by reversed phase LC/MS systems

In this chapter, the focus is on describing and illustrating the procedure on how to obtain high-purity lead compounds for either Ames or short-term toxicology tests using SFC purification A systematic approach to develop a successful SFC purification method is discussed Three different case studies are presented; examples include single-and multistep purifications of typical mixtures, as well as a multistep

purification of a highly complex, impure mixture The advantage of coupling two columns (identical or different) in SFC to enhance separation is also illustrated

A general purification procedure is shown in Scheme 71 The initial steps in Scheme 71 are a standard SFC/MS screen with four chiral columns and a standard

reversed phase LC/MS analysis The strategy is to identify and distinguish achiral and chiral impurities in the original crude sample by utilizing two orthogonal modes of separation (SFC and reversed phase chromatography) Tier-1 screening refers to the initial SFC screening using four chiral columns If a separation is not available with the initial tier-1 screening, 10 additional columns are employed for further method development (tier-2) Once a successful separation method is developed and optimized, scale-up and purification are performed Details of the process are further discussed in Sections 721 through 724 and 73

Tier-1 screening refers to the initial SFC screening using the four columns (46 × 150 mm, 5 μm particle size) listed in Table 71 The most common organic solvents are MeOH, EtOH, and IPOH with or without 02% DEA, depending on compound stability or basicity A generic 6-or 12-minute gradient (5%–50% organic) applies to all tier-1 SFC analytical columns The flow rate is normally 3-5 mL/min, and the oven temperature is typically set at 35°C-40°C The oven temperature and outlet pressure are kept consistent for all gradient analyses

A commonly used C18 column is Phenomenex’s (Phenomenex Inc, Torrance, California) Gemini® (46 × 150 mm, 5 μm particle size) The mobile phase consists of A (01% trifluoroacetic acid [TFA] or 20 mM ammonium acetate [pH 55] in water) and B (01% TFA or 5 mM ammonium acetate in MeCN) A typical reversed phase high-performance liquid chromatography (HPLC) generic gradient starts at 10% B and increases to 90% B over 20 minutes The flow rate is 10 mL/min, and the column is at ambient temperatures The method is usually used to identify achiral impurities greater than 01% at both 215 and 254 nm Occasionally, a different reversed phase method is developed to ensure complete resolution of the product peak from impurities

The reversed phase LC/MS analysis provides information on the number of achiral impurities and their relative percentage peak areas When analyzing the result from SFC/MS screening, the masses observed in LC/MS are used to extract selected ion chromatograms from the SFC total ion current chromatogram Since relatively short gradient times and columns (15 cm compared to 25 cm in purification scale-up) are used in the analytical SFC gradient screening, a partial separation is considered a “hit” when no other options are available If a successful method is identified at this stage, method optimization and scale-up are performed Otherwise,

if desired separation is not available with the initial tier-1 screening, more columns are employed for further method development

The majority of samples can be purified by methods obtained through tier-1 screening; however, there are cases that require more extensive method development Tier-2 may utilize additional columns (see Table 72) and is conveniently conducted overnight The same three solvents (MeOH, EtOH, and IPOH) used in tier 1 may also be utilized in tier 2 Column switching may be performed through an external 12-port valve (VICI International AG, Schenkon, Switzerland), which is connected to the flow path and computer controlled

Polysaccharide-based chiral stationary phases (CSPs), coated (Sepapak®, CelluCoat™, and AmyCoat™) and immobilized (Chiralpak® IA, IB, and IC), are included in the tier-2 screen In addition to the polysaccharide-based CSPs, the synthetic polymer-based P-CAP™ column is also part of the screen A pyridine column is used to obtain the achiral profile of a sample mixture In situations where only achiral impurities are present, achiral columns such as cyano, diol, and unmodified silica (Princeton Chromatography, Inc, Cranbury, New Jersey) are used in place of selected chiral columns in tier 2 The user can choose the columns suitable to their needs as well, and the selection of tier-2 columns continues to evolve as needed

Ideally, a purification method should provide the best separation with the shortest total purification time The best case is when all chiral and achiral impurities are separated from the peak of interest in a single run with a reasonable cycle time In other cases, a number of strategies such as column coupling; multistep purification; and the evaluation of other organic solvents, such as MeCN, n-propanol, and n-butanol, must be utilized to achieve the desired separation

Through tier-1 and tier-2 screening, partial but inadequate separations may be observed If a single column length is not able to provide the desired resolution, columns can be coupled to enhance separations A low column pressure drop in SFC allows column coupling as a common practice The coupled column chemistries can be identical or different and used in combinations such as achiral-chiral or chiral-chiral [13] Coupling of identical columns is a simple way of enhancing resolution; however, coupling of different types of columns may require a significant amount of time in method development to ensure its reproducibility and robustness In such cases, it is generally considered a better strategy to perform multistep purification to avoid potential complications

Multistep purification is necessary for the following two situations: (1) a single method is available but requires a very long purification run time and (2) no single column can provide a robust separation due to the complexity of the sample For Ames samples, complexity often arises from many low-level impurities of diverse retention times and, thus, multistep purification is required As an example of the first situation, it may be necessary to first eliminate the polar impurities to reduce overall purification time when the polarities of the components are quite varied This strategy allows the second step chiral separation to utilize a lower percentage of organic solvent to enhance chiral resolution with a reasonable cycle time Another situation exists when a large number of impurities are present in the sample and multiple columns with varied selectivities are required It is often observed that a good chiral separation is achieved but an achiral impurity coelutes with the desired enantiomer/diastereomer, thus making multistep purification necessary

In addition to the column coupling and multistep purification strategies, other solvents such as MeCN, alcohol-MeCN mixture, n-propanol, and n-butanol have been used in our laboratory to explore different selectivities from those achieved with the first-pass solvents in the standard tier-1 and tier-2 screening conditions [13,14] It is often necessary to adjust the ratios of protic alcohols versus aprotic MeCN to achieve final purification methods

It is convenient to finalize a method on an analytical system before scaling up the preparative SFC Since the tier-1 and tier-2 screenings are conducted using a generic gradient, some preliminary work must be done to obtain an isocratic method, which will later be scaled up During this step, the elution order of many components including achiral/chiral impurities may be altered Isocratic methods are primarily used for purification because of the ability to stack injections

For any analytical SFC method developed, test scale-up is necessary on the preparative columns and instruments Analytical SFC methods are utilized as guidelines for preparative methods in terms of flow rate and chromatographic characteristics such as resolution and selectivity Throughput in preparative chromatography is optimized after considering three major factors: percentage of organic modifier, loading, and cycle time The solubilities of a sample in organic solvents, as well as in supercritical fluids, are also important factors in the scale-up activity, because the typical ratio of the sample to both mobile and stationary phases is much higher in preparative chromatography [14]

It is typical to complete a test run with three to five stacked injections once the percentage of the organic solvent and the sample load amount per injection are determined Any peak disturbances from the stacked injection must be carefully examined, since it may affect ultraviolet (UV)-based collection During the initial scale-up experiment, it is important to observe signs of sample precipitation in the preparative SFC system, which can be possibly due to the difference of solubility between the organic solvent and the SFC mobile phase

After the fraction is collected from the test scale-up run, the chiral purity and achiral purity of the fraction are assessed in analytical SFC and reversed phase HPLC systems, respectively If the tested fractions do not meet the desired purity, then modification of the method is required Once the test scale-up run is successful, the instrument can be run under optimal conditions over an extended period of time When the sample size is particularly large, and the run is expected to go overnight, the instrument is observed closely during daytime operation to ensure that there is no subtle increase in system pressure normally due to sample precipitation within the system Sample loading should be reduced to avoid precipitation in the purification system; sometimes, the percentage of organic solvent can be increased to maximize the solubility under SFC conditions

Achiral and chiral purities are spot-checked during the purification Fractions are then dried down and subsequently subjected to a vacuum oven at 40°C overnight Final chemical purity is measured with the reversed phase HPLC system using the dried sample Chiral purity is checked in the analytical SFC system using the dried sample to confirm that no racemization occurred during the drying process

In this section, three examples of successful SFC purifications, which were performed to generate high-purity lead compounds for screening, non-GLP Ames, or short-term toxicology tests, are presented These examples include single-and multistep purifications of typical mixtures, as well as the multistep purification of a highly complex, impure mixture

A relatively impure sample was submitted for purification to achieve the appropriate purity for the Ames test Reversed phase HPLC analysis showed five closely eluting small impurities Purity of the crude mixture was approximately 80% at UV 215 nm (see Figure 71), and the masses of the impurities were identified using mass spectrometry under electrospray ionization conditions Molecular weight information for the impurities was used to facilitate the development of an analytical SFC method

After the initial SFC/MS screening, an SFC method was identified to separate out all impurities, including the opposite enantiomer As illustrated in Figure 72, the masses of the impurities were extracted to show that the impurities were resolved from the desired peak This was an ideal situation in which all achiral impurities and the chiral impurity could be resolved in a single purification Although the mass (M + 1 = 633) was observed in LC/MS, this peak was not observed in SFC/MS possibly due to sensitivity or because this peak did not elute during the analysis time

It is clear from Figure 72 that the achiral impurities observed in reversed phase LC/MS were resolved in an isocratic SFC method with the AD-H column except the impurity of M + 1 = 633 The peak (M + 1 = 496) at 21 minutes is not a fragment from the target molecule since it has a different retention time in LC/MS The peak (M + 1 = 496) at 31 minutes might be a fragment ion from the target molecule The time interval of 25-35 minutes represents the “window of opportunity” where only the lead compound eluted with no overlap from its enantiomer or other impurities This method was then scaled up on the semi-prep instrument with a flow rate of 60 mL/min with 30% methanol, which provided adequate resolution (see Figure 73) for the 30 mg injection Figure 73 clearly demonstrates that the separation was maintained between the analytical and preparative runs

The collected main product peak fraction was analyzed by reversed phase analytical HPLC and SFC on completion of the purification It was found that chemical purity was greater than 995% (see Figure 74) and chiral purity was greater than 995%

This case study is one of the many examples where single-step SFC purification was sufficient to obtain a high-purity sample when chiral and many achiral impurities were originally present Although a lead compound may be chiral, a chiral column can often separate closely related achiral impurities, as was clearly demonstrated in this example

In this example, 27 g of a crude chiral sample was purified to high purity for the Ames test Initial reversed phase C18 HPLC analysis revealed multiple achiral impurities (see Figure 75) After tier-1 screening, a chiral separation method, using an AD-H column with methanol, showed the presence of a small amount of the opposite enantiomer An isocratic chiral method was then developed to resolve the opposite enantiomer and achiral impurities in the same step During scale-up, however, tailing from the major peak was observed when no DEA was used The typical preparative chromatogram that demonstrates the separation of chiral and several achiral impurities is shown in Figure 76 Analytical chiral supercritical fluid chromatograms are shown in Figure 77

A portion of the achiral impurity at 22 minutes (see Figure 75), however, remained in the collected fraction after the first SFC purification (see Figure 78), requiring additional purification Tier-2 SFC screening was conducted, and the (R,R) P-CAP™ column [15] was found to effectively remove the impurity from the main peak (see Figure 79)

The narrow peak width in addition to the large injection amount (80 mg) with a short cycle time (approximately 2 minutes) allowed the second purification to be completed in a very short period of time Reversed phase analysis (see Figure 710) demonstrated that the impurity observed in the initial purification was completely removed

In this example, a two-step purification was performed to remove both chiral and achiral impurities Initial purification was capable of removing the chiral and most of the achiral impurities The second purification method was rapidly developed due to the reduced sample complexity and the diversity of columns in tier-2 screening Although the achiral impurity in the second purification could also be removed using reversed phase HPLC, SFC provided a shorter run time without using any acid or base additives In addition, the time required to evaporate methanol was significantly less than that would be required to remove the aqueous mobile phase

In this example, two different lots of samples were purified using different methods due to the increased complexity of the second lot An initial lot (10 g) was purified with two OJ-H columns coupled in series (total length was 50 cm) in single-step purification (see Figure 711) A second lot, 40 g of crude sample, was prepared for a short-term toxicological study Unlike the first lot, the second lot was a more complex mixture that contained the opposite enantiomer, diastereomers, and achiral impurities A chromatogram of the reversed phase C18 HPLC analysis is shown in Figure 712

The purity of the main peak based on reversed phase HPLC analysis was 45% at 215 nm (see Figure 712) Because the main peak was a racemic mixture, the content of the desired enantiomer in the crude sample would be less than 23% The sample was analyzed with the tier-1 screen, and two additional pyridine and cyano columns were used to obtain the achiral profile by SFC

A 12-minute-gradient screen was used due to the complexity of the mixture As shown in Figure 713, more components were resolved with OJ-H and AD-H than any other columns studied, and both columns were able to resolve the opposite enantiomer It was determined that the 448-minute peak on OJ-H and the 608-minute peak on AD-H corresponded to the desired enantiomer as confirmed by the injection of the authentic standard The elution order of enantiomers was reversed between AD-H and OJ-H A test run using AD-H/IPOH was chosen as the first step in purification, since the peak at 608 minutes in the AD-H chromatogram (shown in the bottom chromatogram in Figure 713) was most resolved from any other sample components

The preparative SFC chromatogram obtained by a stacked injection of 120 mg of sample is shown in Figure 714 An aliquot of the purified fraction was reinjected onto the preparative SFC system It was found that approximately 10% achiral impurities were still present in the fraction, thus requiring another step of purification

A robust and fast second purification method was found by using an achiral column with a cyano modified functional group An example preparative SFC chromatogram for the second purification is shown in Figure 715 The main peak in the second purification was found to be pure (>995%) after checking the fraction via the reversed phase HPLC method (see Figure 716) as well as the chiral analytical SFC method (see Figure 717)

This example demonstrates that different purification strategies were necessary to purify different lots of the same lead compound Coupling two identical columns enabled a single-step purification of the relatively simple lead candidate mixture

(lot 1) With the known peak of interest from the initial purification, a better strategy was devised for purification of the more complex lot 2 The purification of lot 2 was greatly facilitated due to the ability to reverse the elution order of enantiomers

It is demonstrated in this chapter that high-purity, for example, 97% to up to 995%, lead candidates for both Ames and short-term toxicology tests can be obtained by SFC purification Streamlined method development, such as tier-1 and tier-2 gradient column screening, provides an efficient way to establish a successful SFC purification method Diverse chiral and achiral columns are key components to meet even the most challenging purification requirements A relatively simple scale-up from analytical methods and stacked injections makes efficient SFC purification possible within a reasonable period of time Multistep purification is sometimes necessary to purify a complex mixture by removing chiral and achiral impurities in the most efficient way Coupling two identical or different columns in SFC can enhance separation and provides an additional advantage; it is more readily employed in SFC compared to reversed phase HPLC due to low pressure drop

Overall, SFC is demonstrated to be a very effective purification tool to meet stringent purification requirements Fast method development, quick scale-up, and

column coupling are all attributes critical to the successful use of SFC purification to separate mixtures of various complexities for toxicological studies Additional advantages including reduced organic solvent consumption, together with easy evaporation, make SFC an attractive tool to generate high-purity lead compounds for early toxicological tests