Fast chiral and achiral profiling of compounds with multiple chiral centers by a versatile two-dimensional multicolumn liquid chromatography (LC–mLC) approach

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Fast Chiral and Achiral Profiling of Compounds with Multiple Chiral Centers by a Versatile Two-dimensional Multicolumn Liquid Chromatography (LC-mLC) Approach Jessica Lin writing-original; validation; writing-review& editing; methodology , Charlotte Tsang investigation; visualization; part of writing-original , Raymond Lieu investigation; visualization , Kelly Zhang Ph. D. conceptualization; methodology; supervision; writing-review& editing PII: DOI: Reference:

S0021-9673(20)30185-0 https://doi.org/10.1016/j.chroma.2020.460987 CHROMA 460987

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

18 December 2019 16 February 2020 19 February 2020

Please cite this article as: Jessica Lin writing-original; validation; writing-review& editing; methodology , Charlotte Tsang investigation; visualization; part of writing-original , Raymond Lieu investigation; visualization , Kelly Zhang Ph. D. conceptualization; methodology; supervision; writing-review& editing , Fast Chiral and Achiral Profiling of Compounds with Multiple Chiral Centers by a Versatile Two-dimensional Multicolumn Liquid Chromatography (LC-mLC) Approach, Journal of Chromatography A (2020), doi: https://doi.org/10.1016/j.chroma.2020.460987

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Highlights 

A multiple heart-cutting multiple columns (m) LC-mLC strategy was developed



Separate isomers of compounds with multiple chiral centers and achiral impurities



Simplify multiple chiral center separation into single chiral center separations



Dramatically reduces chiral method development time and sample analysis turnaround



Demonstrated by two case studies of complex pharmaceutical samples

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Fast Chiral and Achiral Profiling of Compounds with Multiple Chiral Centers by a Versatile Two-dimensional Multicolumn Liquid Chromatography (LC-mLC) Approach Jessica Lin, Charlotte Tsang, Raymond Lieu and Kelly Zhang *

Genentech Research and Early Development, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA

*

Corresponding author:

Kelly Zhang, Ph. D. Genentech 1 DNA Way South San Francisco, CA 94080 Tel.: +1 650 467 8470 E-mail: [email protected]

2

Abstract It is critical to determine the chiral impurity profile of pharmaceutical compounds. The rising trend of drug candidates bearing multiple chiral centers has aggravated the analytical challenges. The traditional chiral HPLC methods can take gruelingly long time to develop yet may not offer sufficient resolution for all stereoisomers. A fast analytical strategy with a high success rate is in urgent demand for compounds with multiple chiral centers. In this study, we have developed an effective and fast multiple heart-cutting (MHC) multicolumn two-dimensional liquid chromatography (LC-mLC) platform approach. The m in the name of LC-mLC highlights the employment of multiple chiral columns with different chiral selectors and mobile phases in the second dimension (2D) within the same run, for rapid stereoisomer separation of compounds bearing multiple chiral centers. A short achiral HPLC method in the first dimension (1D) allows the separation of diastereomers and other achiral impurities, followed by 2D analysis enabling different chiral columns and different mobile phases on each coeluted 1D peak for maximum resolution. This LC-mLC strategy breaks down the complex multiplechiral-center separation problems into simple individual one-chiral-center separation, which dramatically reduces chiral method development time and sample analysis turnaround. Its versatile nature and fast turnaround approach have made it a highly efficient strategy to enable quick stereoselective synthetic route development. This platform LC-mLC strategy has been successfully demonstrated in separating eight stereoisomers for a pharmaceutical compound with 3 chiral centers, within total method development time of less than 2 hours and a final analysis time of less than 24 minutes, including column equilibration time. It was also proved highly efficient in separating multiple chiral and achiral components in an in-process sample containing structurally similar starting materials, intermediates, side products and multiple stereoisomers of the product with 3 chiral centers, with minimal method development time.

Keywords: chiral separation, multiple chiral centers, 2D-LC, multiple heart-cutting, multicolumn, chiral impurity

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1. Introduction Although having the same chemical composition and connectivity, chiral stereoisomers may exhibit significant differences in pharmacological properties such as toxicity, pharmacokinetics, and drug metabolism [1, 2]. It is critical to analyze and control the chiral purity of the drugs to ensure delivery of safe and efficacious medicines to the patients. However, chiral separation is often a substantial analytical challenge due to the profound structural similarity of stereoisomers. Furthermore, there has been a rising trend of APIs with multiple chiral centers [3, 4] and the increasing analytical challenge accompanying such a trend, as the number of stereoisomers increases exponentially with the number of chiral centers (2n rule). The most common approaches for chiral separation include high performance liquid chromatography (HPLC), gas chromatography (GC), supercritical fluid chromatography (SFC) and capillary electrophoresis (CE). HPLC is the most widely used among these techniques [5-9]. Chiral separation is typically achieved on the chiral stationary phases (CSPs) including polymer-based carbohydrates, protein-based, Pirkle type, cyclodextrins and chirobiotic type etc. [10]. The separation mechanism on CSP remains less understood, and the chiral HPLC method development is typically less predictable than the achiral counterpart. A number of studies have been published to resolve chiral APIs with two or more chiral centers [3, 4, 11-13]. Hassan and coworkers developed a chiral HPLC method to separate nebivolol which has four chiral centers, and herein a total of 10 stereoisomers due to restriction from plane of symmetry. All the ten stereoisomers of nebivolol were separated under isocratic conditions using CHIRALPAK AD-H column with a total analysis time of 120 minutes [11]. Another study separated Aprepitant with three chiral centers, where eight stereoisomers were resolved by using CHIRALPAK AD-H column with a total chromatographic time of 70 min [12]. In both cases, tremendous method development time and effort have been invested in addition to the long methods, which greatly limited the efficiency of analysis. Furthermore, in many situations the resolution of all stereoisomers may not be possible with a single chiral column even with extensive method development.

4

Enantioselective synthesis is a common strategy for chirality control in pharmaceutical industry, and the synthetic development relies on the chiral characterization of the process intermediates. The turnaround and quality of analytical data are critical to drive the timeline of route selection and optimization. The aforementioned lengthy yet less successful chiral method development practice does not align with such demands, where the analytical methods have to be developed quickly to fit in the project timeline and to be adjusted swiftly to adapt to the synthetic route change. There is a huge unmet need for a fast, flexible and versatile methodology with minimum method development to characterize the chiral profile of pharmaceutical materials, especially in the discovery and early development stage. 2D-LC can effectively increase the peak capacity and resolving power compared to traditional 1D-LC by combining different chromatographic columns with orthogonal or complementary separation features [14-16]. It has been successfully utilized in challenging pharmaceutical chiral separation [17-20]. Typically, the first dimension (1D) uses an achiral column, where the stereoisomers of interest are isolated from the other achiral components and then separately transferred to the second dimension (2D) for chiral separation. When separating compounds with multiple chiral centers, coelution may occur to more than one peak in the 1D, necessitating multiple heart-cutting (MHC) to transfer multiple coeluting stereoisomers to the 2D. All of the published LC-LC studies to our knowledge use a single stationary phase in the 2D to analyze each 1D fraction, and some applications only achieved partial resolution for some fractions. It is not surprising since a single chiral stationary phase typically does not provide sufficient resolution power for all stereoisomers. In this study we have developed a LC-mLC 2D-LC approach which utilizes multiple chiral stationary phases and mobile phases in the 2D to resolve stereoisomers of compounds with multiple chiral centers and related achiral impurities. The m in LC-mLC means that the system has the capability of screening multiple stationary phases and mobile phases in the 2D as well as the flexibility of implementing multiple stationary phase and mobile phase in the 2D for different 1D fractions within the same run. Compared to conventional 1D-LC methods or LC-LC methods that use a single

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stationary phase in the 2D, this methodology offers superior chromatography resolution and development efficiency due to the increased selectivity and orthogonality. It essentially simplifies the complex multiple chiral center issue into a simpler single chiral center separation. The first step of the methodology is to separate diastereomers and other achiral components with a fast achiral method in the 1D. The second step is to transfer the peaks of interest from the 1D to the 2D for automated column and mobile phase screening. The analyst may stop the column screening whenever satisfying resolution is achieved for all MHC fractions and characterize the chiral profile “on the fly” from the screening result, without further method optimization or re-analyze the sample. If better resolution or faster analysis is needed, the third step can be pursued to quickly optimize the methods in the 2D. Two case studies of a Genentech pharmaceutical compound with three chiral centers and an in-process sample with three chiral centers and multiple achiral impurities will be discussed here to exemplify this strategy. The methodology has been demonstrated

as

a

versatile,

efficient

and

successful

generic

chiral-achiral

characterization tool for compounds of multiple chiral centers to support enantioselective process development, especially in the discovery and early development stage.

2. Experimental 2.1. Reagents and Materials Formic acid (98-100%) and acetic acid (100%) were purchased from EMD Millipore (Billerica, MA, USA). Acetonitrile (HPLC grade) was from J.T Baker (Center Valley, PA, USA). Ethanol, hexane and methanol (all HPLC grade) were from EMD Millipore (Billerica, MA, USA). Ammonium formate was from Sigma-Aldrich (St. Louis, MO, USA). De-ionized water was from Milli-Q water purification system (Millipore, Bedford, MA, USA). The Genentech pharmaceutical intermediate and its stereoisomers, and the in-process sample were synthesized in-house.

2.2. Instrumentation

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The 1D includes a tertiary gradient pump, an autosampler, a thermostatic column compartment, and a diode array UV detector. The 2D includes a tertiary gradient pump, a thermostatic column compartment with a six-position switching valve, a UV diode array detector, and a Thermo LCQ Fleet mass spectrometer (San Jose, CA, USA). All HPLC compartments were Model Ultimate 3000 purchased from Thermo Fisher Scientific (Sunnyvale, CA, USA). Channel A of the tertiary pump in the 2D was connected to a tenposition solvent switching valve for mobile phase screening. The interface between the 1

D and the 2D is a Rheodyne six-loop valve trapping system (Oak Harbor, WA, USA)

with the sample loop volume of 50 µL. Chromeleon 6.8 Chromatography Management Software was used for system control, data acquisition and data processing.

2.3. Chromatographic Conditions for the Case Studies 2.3.1 Case Study I In the one dimensional chiral HPLC method development for the Genentech compound in the case study I, eight polysaccharide columns including CHIRALCEL OD-3, OJ-3 and CHIRALPAK AD-3, AS-3, IA-3, IG-3, IE-3 and IC-3 (Chiraltech, all 4.6 mm × 150 mm, 5.0 µm) were screened and optimized. The final optimized method utilized CHIRALPAK IA (4.6 mm × 150 mm, 5.0 µm) running gradient from 30 to 70% MPB in 20 minutes, where MPA was 0.1% formic acid in ethanol and MPB was hexane. The column temperature was 30 oC and the flow rate was 1 mL/min. UV wavelength was set at 280 nm. For the two dimensional study, the 1D separation was carried out on a Sigma-Aldrich Ascentis Express C18 column (Sigma Aldrich, 4.6 mm × 150 mm, 2.7 µm). Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in methanol. Isocratic separation was performed at MPA/MPB 25%/75% for 12 minutes. The flow rate was 1.0 mL/min. UV detection was performed at 280 nm. For the 2D screening, each of the four 1D peak was trapped into a 50 µL stainless steel loop by time-triggered collection, and screened with six polysaccharide columns in the order of CHIRALCEL OD-3, CHIRALPAK IG-3, CHIRALPAK AS-3, CHIRALCEL

7

OJ-3, CHIRALPAK IA-3 and CHIRALPAK IC-3 (Chiraltech, all 3.0 mm × 50 mm, 3.0 µm) in the 2D. Mobile phases A was 10 mM ammonium formate at pH 3.7 in water, and mobile phase B was acetonitrile. The gradient employed was mobile phase B from 60 to 90% in 4 min with 1-min equilibration at 60%. The flow rate was 1.0 mL/min. The injection volume was 2 µL. UV detection was performed at 280 nm. For the final optimized 2D condition, CHIRALCEL OD-3 was selected for Peak 1 and 4, and CHIRALPAK AS-3 for Peak 2 and 3. The 2D method was isocratic at MPA/MPB 20%/80% for 3 minutes. The mobile phases, flow rate, UV wavelength and column temperature were the same as in the 2D screening.

2.3.2 Case Study II The 1D separation was carried out on an Agilent Poroshell EC-C18 column (3.0 mm × 150 mm, 2.7 µm). Mobile phase A was 10 mM ammonium acetate pH 4.8 in water and mobile phase B was ACN. The gradient employed was mobile phase B from 20 to 70% in 3 min, then from 70 to 95% in 9 min. The flow rate was 0.3 mL/min. UV detection was performed at 280 nm. For the 2D screening and analysis, the same strategy in 2.3.1 was used, with the same trapping loop size, the same columns and gradient, flow rate and UV detection wavelength, except that the mobile phase A was 20 mM ammonium acetate pH 9 in water, following the LC-mLC strategy described later in section 3.1.

2.4. Sample Preparation All samples were prepared with ethanol in class A glass volumetric flasks. For Case Study I, the tertiary alcohol intermediate was enriched to a sync-mixture (containing RSS, SRR, SRS and RSR, in theoretical ratio of 1:1:1:1, Mixture 1) and an anti-mixture (containing SSR, RRS, SSS and RRR, in theoretical ratio of 1:1:1:1, Mixture 2) respectively. Mixture 1 and Mixture 2 were both prepared at 1 mg/mL separately. The method development sample was prepared by mixing Mixture 1 with Mixture 2 in 1:1

8

(w/w) ratio. The sensitivity sample was prepared by spiking 0.05% (w/w%) of Mixture 2 to Mixture 1. For Case Study II, the samples were dissolved in 50/50 acetonitrile/water.

3. Results and Discussion 3.1 Design of the LC-mLC Strategy The platform LC-mLC strategy combines a single achiral column in the 1D and multiple chiral columns in the 2D, to achieve superior selectivity and peak capacity compared to the conventional 1D chiral LC methodology or the 2D achiral-chiral LC-LC strategy using a single chiral column. The main goal of the 1D achiral method in the LC-mLC strategy is to resolve diastereomers and other potential achiral impurities, leaving the resolution of each enantiomer pair and any other coelutions with achiral impurities to the 2

D mLC chiral methods. When we approach separation of a complex stereoisomer

mixture in such a stepwise manner utilizing different column chemistries in the 1D and the 2D, we alleviate the resolution requirement on each dimension of chromatography. Six polysaccharide columns including OD-3, IG-3, AS-3, OJ-3, IA-3, and IC-3 are screened in the 2D. The columns are selected because they have the highest success rate based on our experience, literature and column vendor’s suggestions. The 2D chiral screening is conducted in the reversed phase mode, to minimize the solvent incompatibility between the 1D and the 2D. Two mobile phases A, 10 mM ammonium formate at pH 3.7 (for acidic or neutral compounds) and 20 mM ammonium acetate at pH 9 (for basic or neutral compounds) in water were set for the generic screening. Mobile phase B was defaulted to acetonitrile but other solvents can also be screened. Gradient screening is employed in the 2D. To maximize the screening success in the 2D, the gradient slope can be adjusted to 10-70% for more hydrophilic compounds, or 30-90% for the general compounds and 60-90% for the more hydrophobic ones, based on the retention behavior from the 1D reversed phase separation. It shall be noted that the analyst does not have to complete the whole screening, and can stop anytime when the appropriate columns and mobile phase to separate all the peaks of interest from the 1D are identified.

9

The strategy stated in this article has four major highlights. Firstly, the presented system and strategy allow different columns and different mobile phases to be used in the 2D for different MHC fractions in the same 2D-LC run. This is enabled by implementing a column-switching valve in the 2D column compartment, a solvent-switching valve in the 2D pump, and customized programming for instrument control as shown in Figure 1. Most published 2D-LC work only use one stationary phase in the 2D to our knowledge, and the analysts are bound to a single 2D method to resolve the coeluting species in each fraction, which in many cases cannot be achieved even with substantial method development effort. With the presented strategy here, we have decoupled the dependence of each fraction, and the analysts are not obliged to find a single “one size fits all” column to resolve all MHC components. Rather, they have the flexibility to select different columns for individual MHC fractions. Similarly, different mobile phases (either aqueous phase or organic phase) can be used for different MHC fractions for enhanced resolution when necessary. This has markedly reduced the method development demand and improved the chance of baseline resolution of all fractions. Secondly, column and mobile phase screening and selection can be automated in the 2D. In all the published 2D-LC work to our knowledge, the 2D column selection and method development are either done on a different HPLC system with a multi-column chamber or on the same 2D-LC system by manually switching columns in the 2D [18]. The presented LC-mLC system in this study symbolizes in the column screening and mobile phase screening capability in the 2D with implementation of a 6-position column switching valve (CSV) and a 10-position solvent switching valve (SSV). This setup allows obtaining final sample analysis result “on the fly” from the method screening, without having to rerun the sample by a final selected method, which significantly streamlines the 2D method development and eliminates the need of method transfer between systems. Thirdly, this approach enables unambiguous peak tracking. It addresses the peak tracking issue where individual compound markers are not available for method development, which is common in early stage of drug research and development. With chiral

10

compounds, the traditional method development approach is to conduct both chiral and achiral column screening in parallel to get the achiral and chiral impurity profiles separately. One significant caveat of this approach is the peak tracking between methods without the individual retention markers, especially with diastereomers which may be resolved by both achiral and chiral methods but cannot be distinguished by UV or mass spectrometry detection. The presented strategy integrates achiral and chiral methods in the same analysis, transfers individual peaks from the 1D achiral separation for further 2D chiral separation, thus enables unambiguous peak tracking. Lastly, the speed and flexibility of this approach have distinct advantages to be utilized as a generic methodology in drug discovery and early development stages, where a fast chiral characterization is needed for fast decision making, but a fully validated analytical method is not yet necessary. It will be demonstrated in details in the following Case Study section how this strategy provides fast characterization of the challenging stereoisomer impurity profile with minimal method development effort.

3.2 Case Study I 3.2.1 Highly Stereo defined Alcohol Intermediate The compound of interest, a Genentech process intermediate is a tertiary alcohol. As shown in Figure 2, three chiral centers are present with eight potential stereoisomers. The syn-tertiary alcohols are desired to form E-olefin in the downstream process, while the anti-tertiary alcohols lead to undesired Z-olefin. An analytical method is needed to demonstrate the chiral purity of the alcohol intermediates and stereoselectivity of the coupling steps, and to ensure the correct configuration of the olefin product. The analytical method shall resolve all 8 stereoisomers of the tertiary alcohol intermediate.

3.2.2 Conventional 1D-LC Method Development A conventional one dimensional LC method development approach was first attempted to separate these 8 stereoisomers. An extensive chiral HPLC method screening was conducted, aiming to separate all 8 stereoisomers using one method. There are a plethora

11

of studies on chiral screening strategy and the discussion of such strategy is not the focus of this article. Based on the existing literature[21, 22], our past experience [23] and recommendations from column manufactures, we conducted a comprehensive chiral HPLC screening with chiral stationary phases including CHIRALPAK IA, IE, IC, IG, AD, AS, and CHIRALCEL OD, OJ. Reversed-phase, normal phase and polar ionic phase conditions were screened extensively. The best separations from column IA, AD and OD were further optimized by adjusting the gradient or isocratic conditions, mobile phases and additives, column dimension, flow rate and temperature. However, the resolution was not significantly improved. After optimization, the best condition giving the highest number of resolved peaks was obtained with CHIRALPAK IA, where 5 isomers were baseline resolved and 3 isomers coeluted as shown in Figure 3. The whole method development and optimization took more than a month, yet no baseline resolution could be achieved for all components. Although the chiral recognition mechanism is not fully understood, there are two possible reasons to explain the challenge of separating compounds with multiple chiral centers. Thermodynamically, the configuration and interaction capability of chiral binding sites on a single CSP are rather limited, and may not offer sufficient differential Gibbs free energy changes for stereoisomers at every chiral center simultaneously. Hence chromatography selectivity α may be high at some chiral centers, but low at the others. The likelihood of full resolution for all stereoisomers decreases as the number of chiral centers

increases,

and

this

can

be

explained

statistically.

Kinetically,

the

desorption/adsorption rate constant of compounds in a chiral binding process is intrinsically lower than achiral interaction [24]. Therefore the efficiency and peak capacity are lower with chiral HPLC compared to achiral HPLC, leading to lower resolution which is more detrimental to compounds with multiple chiral centers. The conventional chiral 1D-LC screening strategy has consumed tremendous effort but yielded limited success, and we targeted to overcome such limitation with the new LCmLC approach presented here.

3.2.3 LC-mLC Strategy

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To analyze the alcohol intermediate with the strategy, the 1D method was adjusted from the intermediate achiral HPLC method and four peaks were observed as in Figure 4. The first two peaks are SRS/RSR and SRR/RSS or SRR/RSS and SRS/RSR, whereas the last two peaks are SSS/RRR and SSR/RRS or SSR/RRS and SSS/RRR, based on the identity of the two enriched sync- and anti- mixtures, and the fact that enantiomers cannot be separated in the achiral environment. In each chiral screening run in the 2D, four peaks were collected in four separate sample loops and sequentially sent to a single column in the 2D for further separation. Because of the acidic nature of the compound, a single mobile phase A at pH 3.7 was used. The column screening sequence was set up in the order of OD-3, IG-3, AS-3, followed by OJ-3, IA-3 and IC-3 based on our experience of column performance ranking. Up to 6 screening runs can be conducted to go through all columns and the runs can be programmed in a single screening sequence in Chromeleon. The analyst can obtain the screening results on the fly and can stop the screening whenever satisfying resolution is achieved. The column that provided the best resolution was selected for each pair of enantiomers. AS selected for Peak 2 and 3, and OD was selected for Peak 1 and 4 as in Figure 4. It shall be noted that neither AS nor OD alone can resolve the enantiomers of all four peaks from 1D. An LC-LC approach with AS in the 2D would only have achieved partial resolution of the enantiomers of Peak 1 and 4, and an LC-LC approach with OD in the 2D would have coelutions of enantiomers of Peak 2 and 3. Hence the traditional LC-LC methodology was not effective in this situation. The capability to use different columns in the 2D (LC-mLC) within the same analysis is a key innovation in this methodology compared to the typical LC-LC approach. It has broken the interdependence of 1D fractionations and the choice of the 2D columns so that any 1D fraction can go to any 2D column, independent of other fractions. It has significantly improved the separation orthogonality, usage of separation space and therefore overall resolution. The resulted significant merit is the efficiency. Because resolution of all 4 fractions was achieved with the first three columns screened, the screening was stopped there, resulting to a total screening time of 1.6 hours. If the analyst chose to complete the whole screening, it would have taken 3.2 hours. The data obtained from the hits during the screening process can be directly used for chiral profiling, but more optimization can be done if a method is 13

needed to characterize more synthetic samples. In this case, the 2D methods were quickly optimized by changing to a 3-minute isocratic condition to improve the resolution and reduce the time for column equilibration. The final 2D chromatograms are shown in Figure 5. The final method was only 24 minutes long, with separation of all eight stereoisomers, and such resolution cannot be obtained with a single column even with substantial method development effort as discussed in Section 3.2.2. The fast HPLC analysis time has provided the necessary speed for stereoisomer characterization, on which the process chemists have relied to drive the synthetic route development. Furthermore, the whole method development to deliver the 2D-LC successful separation took less than 2 hours, making the strategy highly suitable in the fast-paced pharmaceutical discovery and early development environment. The synthetic routes at this phase are prone to significant changes; hence the analytical methodology has to be fast and versatile.

3.2.4 Quantitative Analysis It has been known that the 2D may experience resolution loss due to solvent strength mismatch between the 1D and 2D, especially when a large sample volume is transferred to the 2D [25, 26]. Therefore, a sample fraction of 50 µL was collected to minimize solvent incompatibility and peak distortion in the 2D. It should be noted that although only the apex of the 1D peak has been sampled, it does not impact the ratios of enantiomers in the 2

D because the enantiomers totally coelute on achiral column in the 1D, and ratio of

enantiomers stays the same across the peak. The LC area percent of each stereoisomer can be calculated as:

where Area1D% is the peak area percent of the specific stereoisomer and its coeluting enantiomer in the 1D, and Area2D% is the peak area percent of the specific stereoisomer relative to its enantiomer in the corresponding 2 D run. For instance of the sample in Figure 4, the area% of the eight stereoisomers are 12.37% (24.69% × 50.09% × 100), 12.32% (24.69% × 49.91% × 100), 13.12% (26.13% × 50.22% × 100), 13.01% (26.13%

14

× 49.78% × 100), 11.95% (23.37% × 51.15% × 100), 11.42% (23.37% × 48.85% × 100), 12.52% (25.82% × 48.50% × 100) and 13.30% (25.82% × 51.50% × 100) respectively, and the total adds up to 100%.

3.2.5 Sensitivity, Specificity and Repeatability According to ICH Q3A, a pharmaceutical impurity has a reporting threshold of 0.05%, an identification threshold of 0.10% and a qualification threshold of 0.15% for daily dosage of 2 g or lower. While sensitivity is an important attribute of an analytical method for pharmaceuticals, it has been less studied with 2D-LC [27, 28]. The sensitivity of the methodology was demonstrated by spiking the Mixture 2 at 0.05% of the concentration of Mixture 1, with each individual stereoisomer at 0.0125%. The Peaks 3 and 4 were sampled and transferred to the 2D and S/N values were achieved in the range of 10 to 20 as shown in Figure 6 (black traces in bottom figures). The strategy is sensitive enough to detect chiral impurities down to 0.0125%. A diluent was injected in 1D, and four fractions were collected at the same time windows as the sample. No interference peaks were observed from the diluent run in the 2D (blue traces in Figure 6), demonstrating the method specificity. Table 1 Repeatability of the quantitative results Enantiomer

Enantiomer

Enantiomer

Enantiomer

Pair 1

Pair 2

Pair 3

Pair 4

24.69

26.13

23.37

25.82

%RSD of D %Area

0.12

0.45

0.41

0.44

2

50.09

50.22

51.15

48.50

0.46

0.28

0.65

0.55

1

D Avg. %Area 1

D Avg. %Area 2

%RSD of D %Area 1

D Avg. %Area: peak area percent of the enantiomer pair in the 1D chromatogram

2

D Avg. %Area: peak area percent of the first-eluting enantiomer in each 2D chromatogram

The repeatability of the method was demonstrated by 6 repeated injections of the same sample and obtaining the %RSD of the %Area for each stereoisomer as shown in Table 1. Because each pair of enantiomers was resolved in a separate chromatogram in the 2D, and the 2D %Area of one enantiomer and the other added up to 100%, it is only necessary to

15

calculate the %RSD of the %Area for the earlier eluting enantiomer for the 2D repeatability assessment. The %RSD of both the 1D and 2D %Area was less than 1%, indicating great repeatability for quantitation. In summary, the methodology has demonstrated excellent sensitivity, specificity and repeatability for chiral profiling and quantitation. We took a stage appropriate method qualification here to demonstrate the method is appropriate for discovery and early development stage for fast process development decision making. For quality control purpose and late stage development where method transfer is expected, method robustness such as lab-to-lab, column-tocolumn and day-to-day performance should be further evaluated. The robustness of 2DLC method in quality control and late stage development setting was thoroughly evaluated in our previous work [28].

3.3 Case Study II In-process samples are typically complex in composition, containing the starting materials, intermediates, products, byproducts and other impurities. The presence of multiple chiral centers can only add to the complexity, as both chiral and achiral methods have to be developed, and the results of the two individual methods have to be harmonized. The LC-mLC methodology can be extremely helpful in this situation to probe the maximal impurity information. In this case study, we applied the methodology to characterize a complex in-process sample where both achiral and chiral components were present. In a telescoped two-step process discussed here, the first step is the reduction of a ketone starting material (SM) to an alcohol, which can lead to the desired S-alcohol (S1) and undesired R-alcohol (R1); along with the S and R forms of the structurally similar ring-opening side products (S2 and R2). The second step is the hydrogenation of S1 intermediate which produces two additional chiral centers and a total of 5 stereoisomers (RRS, SSS, SSR, SRS and RSS) based on the asymmetric synthesis, with SSR being the desired configuration. An inprocess sample after Step 2 can potentially contain all the 10 components, which are either stereoisomers or structurally similar non-stereoisomers, and some other process impurities, and resolution of all components was to be pursued.

16

A composed reaction mixture was used for method development. A typical reservedphase method (see details in the Experimental section) was used to in the 1D to resolve diastereomers and some other achiral impurities. As shown in Fig. 7, 8 peaks were resolved within 8.5 minutes. Based on the MS information and available markers, Peak 1, 3 and 4 are stereoisomers of the Step 2 product, Peak 2 is the Step 1 ring-opening side products (S2+R2) and Peak 5 is the Step 1 product (S1) coeluatd with several impurities. Two low-level unknown impurities were also observed. Because of the basic nature of the compound, the basic mobile phase A was used following the strategy in section 3.1. Each of the 5 peaks in the 1D was heart-cut and screened on the six chiral columns in the 2

D, and the column that resolved the most components in the 2D was selected as the final

method for that fraction as shown in Figure 7. Note that four different chiral columns including IA (for Peak 1), IC (for Peak 2), IG (for Peak 3 and 5) and AS (for Peak 4) were selected for maximal 2D resolution, which again demonstrated the flexibility of the methodology that multiple columns can be used in 2D for a single sample injection. Other than the stereoisomers, additional achiral impurities were resolved for each 1D peak by the 2D chiral columns. In total, 8 additional intermediate related impurities (IM1, IM2, IM3, IM4, IM5, IM6, IM7 and IM8) were resolved in the 2D. By combining 1D and 2D separation, we were able to resolve the 20 components, both chiral and achiral, in the inprocess sample within a 37-minute total run time. For quantitative information, the area% of each component of interest was calculated as in the equation in section 3.2.4. For example, the %area of the desired Step 2 SSR product is 1.41% (10.04% × 14.07% × 100) and the %area of the desired Step 1 S1 product is 12.35% (34.16% × 36.16% × 100). Neither the conventional 1D-LC approach nor LC-LC approach could offer the same resolution and speed. The 1D-LC approach would take separate achiral and chiral method development and the peak tracking between the achiral and chiral method would be challenging due to the complexity of the sample composition. Furthermore the resolving power can be rather limited by combining a single achiral method and a single chiral method. While the peak tracking can be solved by integrating the chiral and achiral method with LC-LC, the resolution is still far less optimal from a single 2D chiral column for all of the components. As apparent from this case study, 4 different chiral columns were used to maximize the resolution of each individual 1D fraction in the 2D. Some 17

stereoisomers and impurities would have been missed if only one column was used with conventional LC-LC approach. More importantly, in this case study, the characterization data was obtained “on the fly” without further method refinement in the process of 2D column screening, where the turnaround is highly suitable for fast analysis. This strategy is highly valuable in providing fast and comprehensive characterization of in-process samples for fast process development.

4. Conclusion In this study we have developed a versatile two dimensional multiple heart-cutting multicolumn LC-mLC approach as a generic methodology for separation of stereoisomers with multiple chiral centers. The muticolumn (m) 2D-LC methodology has combined an achiral column in the 1D and multiple chiral columns and mobile phases in the 2D to maximize chromatography resolution and minimize method development time. The strategy has been proven successful in characterizing the chiral impurity profile of a complex pharmaceutical molecule with 3 chiral centers, with excellent sensitivity, specificity and repeatability. The method was developed in less than 2 hours, and complete resolution has been achieved for all 8 stereoisomers in 24-min run time, while extensive traditional 1D-LC method development cannot provide matching performance. The strategy was also applied in characterizing both chiral and achiral components “on the fly” in an in-process sample. The strategy highlights superior resolution and fast turnaround, which makes it a highly efficient generic analytical strategy for quick enantioselective process development and control in the fast-paced pharmaceutical environment. Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author statement

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Jessica Lin: writing-original, validation, writing-review& editing, methodology Charlotte Tsang: investigation, visualization, part of writing-original Raymond Lieu: investigation, visualization Kelly Zhang: conceptualization, methodology, supervision, writing-review& editing

Captions

Fig. 1 Setup of the MHC LC-mLC system. The system is capable of trapping up to 6 peaks from the 1D in a single run. The 2D column chamber accommodates 6 columns. The fractions from the 1D can go to different 2D columns in a single sample injection.

Fig. 2 Structure of the tetrasubstituted alcohol chiral isomers in Case Study I.

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Fig. 3 The chromatogram of the best one dimensional separation of the tetrasubstituted alcohol chiral isomers, where 5 stereoisomers were resolved from a total of 8 and three stereoisomers coeluted. Peaks 2, 3, 5 and 7 were from anti-mixture (containing SSR, RRS, SSS and RRR), Peaks 1, 4, 6, 8 were from sync-mixture (containing RSS, SRR, SRS and RSR). See Experimental Section for detailed chromatographic conditions.

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Fig. 4 The LC-mLC 2D screening of each 1D fraction of the tetrasubstituted alcohol chiral isomers with CHIRALPAK AS and CHIRALCEL OD columns . See Experimental Section for detailed chromatographic conditions. The chromatograms in the red circles are selected as the final methods for each pair of enantiomers and used for final data reporting.

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Fig. 5 The LC-mLC chromatograms of the separation of the tetrasubstituted alcohol chiral isomers with three chiral centers with a total run time of less than 24 mins . See Experimental Section for detailed chromatographic conditions.

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Fig. 6 Sensitivity and specificity results with 0.05% spike of Mixture 2 into Mixture 1. Peak 3 and 4 (top) were sampled into the 2D and the resolved peaks (black traces in the bottom figures) showed signal-to-noise ratio (S/N) over 10. The 2D chromatograms of diluent (blank) injection showed no interference at the respective retentions times (blue traces in bottom figures).

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Fig. 7 The LC-mLC chromatograms of the separation of an in-process sample involving three chiral centers and multiple structurally similar achiral impurities. See Experimental Section for detailed chromatographic conditions.

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