Analytical Method Lifecyle of SFC Methods from Development Use to Routine QC Implementation; Supporting Small Molecule R&D and Commercialization

Selection of the correct analytical technique from the outset of method development is key in securing the optimal and most robust method to be used throughout a product’s development journey from R&D to commercialization. In this article, we review some key considerations for chromatographic technique selection and method development across the full drug process—from early-stage API synthesis to routine commercial release activities. We describe the implementation of supercritical fluid chromatography (SFC) in the pharmaceutical industry, especially for chiral, water sensitive analytes, and low to high LogP and LogD hydrophobic compounds, while demonstrating high levels of method robustness and support of “green” analytics.

The product development lifecycle in the pharmaceutical industry can be divided into three distinct phases: discovery, development, and registration and manufacturing.

In the discovery phase, the analytical impetus is normally on speed and throughput. However, once a candidate has passed the proof-of-concept stage selected for development and used in clinical trials, the analytical methodology that accompanies the development should take into consideration the overall method lifecycle, which may extend through to routine product manufacturing support. Such analytical methods need to meet requirements of United States Pharmacopeia (USP) resolution (USP <621>) (1–2) and sensitivity (limit of quantification [LOQ] and limit of detection [LOD]). Eventually, the full development to registration and manufacturing has to guarantee robustness and ruggedness, ensuring good transferability of the method. Meeting sustainability goals is also a key driver that should be considered, as these analytical methods could be used for a considerable period of time (in the order of years).

The required performance of the analytical methodology, defined in the product analytical target profile (ATP) serves as the starting point for method technique selection and method development (3). The correct marrying of “method requirement,” “analyte properties,” and “technique capability” is what ultimately ensures robustness of the method. Specificity, sensitivity, accuracy, and reproducibility are typical parameters that measure the performance of the analytical method. The technique should be able to meet these performance criteria versus the analyte, instrumentation and transferability across laboratories, departments and business units.

Prior knowledge or experimental verification relating to the performance of available analytical technologies enables the analyst to select the most appropriate technology to meet the requirements of the ATP. Where more than one technology has been demonstrated to meet the ATP, a review of business requirements (such as throughput, automation, downstream availability) should be performed to aid selection. Procedure specific performance indicators should be defined. These can include critical resolution of defined impurities (also known as the key predictive sample set [KPSS]), as well as specific sensitivity requirements, and this becomes the starting point of method development.

Discussion

The choice of analytical technique plays a critical role in the success of the analytical method. The key requirements from the method are defined in the ATP, and the analytical technology needs to be able to satisfy all these requirements–not only during the development, but also throughout the project lifecycle, from development to commercialization. If a non-robust or poorly optimized method is developed in the first instance, subsequent method redevelopment will be needed downstream, generating a cumulative resource burden.

In the pharmaceutical industry, reversed-phase liquid chromatography (RPLC) is by far the most widely employed analytical technique, and carries several advantages over alternative techniques, including analyst familiarity and versatility for many different molecular classes. Although alternative separation modes are often proposed during development, technique selection is often driven by business considerations, such as instrument availability across downstream or partner laboratories. In agreement with ICH-Q14 (4), other techniques should be prioritized for method development if they provide higher method robustness, instrument ruggedness, validation success, and transferability.

While the analyst can rely on the availability of a wide toolbox of separation techniques (including, but not limited to, RPLC, supercritical fluid chromatography [SFC], gas chromatography [GC], ion exchange [IEX] chromatography, capillary electrophoresis [CE], hydrophilic-interaction chromatography [HILIC], normal phase liquid chromatography [NPLC], and size-exclusion chromatography [SEC]), each of them can perform optimally only within defined constraints of physicochemical properties, such as pK_a, logD, logP, or solubility. Each chromatographic mode has an operating “sweet spot,” and, within this sweet spot, the optimal performance and robustness for the analytical method is unlocked. In this article. we discuss the application of SFC identified as the optimal approach to meet ATP requirements in various measurement challenges.

The Road of Enabling SFC from Development to QC

As discussed earlier, SFC may be the first choice for analytes that have compatible physicochemical “sweet spots,” for example:

Retention:

• Analysis of polar compounds, (LogD < -1): While SFC provides adequate retention, RPLC risks eluting such compounds with the solvent front unless appropriate fully aqueous-compatible stationary phases are selected or ion pairing reagents utilized. This leads to additional method complexity, and often require additional method controls.
• Hydrophobic compounds (LogD > 4): With RPLC, such analytes are typically eluted later in the gradient… or not at all.

Solubility:

• SFC is a good technique for the analysis of lipophilic analytes with high solubility in organic solvents (and poor solubility in water). Selecting RPLC for such an application elicits a risk of precipitation (column blockage), diluent or mobile phase incompatibility (potentially leading to poor peak shapes), insufficient column loading, or carry over.

Stability:

• The “water-free” mobile phases employed with SFC offer the advantage of being the ideal choice for the analysis of water labile compounds.

Selectivity:

• SFC has useful orthogonality (chromatographic selectivity) to RPLC. This alternative orthogonality has been shown to offer superior positional isomer selectivity, which can be useful in resolving difficult critical pairs (4).
• SFC is Pfizer’s (and many other companies) default choice for chiral separations.

The Use of SFC–MS in an Open Access Environment

In our laboratories, an open access (OA) SFC–MS instrument has been in use since 2021 by both chemists and analysts. The availability of easy-to-use generic method screens (chiral and achiral) facilitate high throughput screening of a large number of process chemistry samples.

In 2022, over 3000 samples were analyzed on this open access SFC. The OA SFC screen has significantly decreased method development time, and, by incorporating SFC earlier on in the development workflow (at the initial process chemistry stage), has led to the organic growth of the technique in the department. The inclusion of an open access SFC has therefore lowered the energy-barrier for wider SFC application in development at Pfizer.

SFC for Drug Product Development

Due to its applicability to analytes with low solubility in aqueous environments, SFC also has application in drug product (DP) analysis. The application detailed here is for an injectable DP that required an oil-based formulation. Castor oil, soybean oil, sesame oil, and oleic acid were explored by the formulation team as potential excipients for an injectable solution of an Active Pharmaceutical Ingredient (API). A risk assessment of a potential RPLC method highlighted several risks, such as the accumulation of the oil matrix or risk of precipitation of the API on column, the risk of inaccurate analyte recovery, intensive sample preparation, tighter system control requirements, and higher maintenance burden. With SFC being a “water-free” chromatographic technique, and with the API and formulation oils being soluble in organic solvent and CO₂ mobile phases, most of these risks were mitigated. On a polar stationary phase, the API peak eluted in the middle of a 2% to 42% organic modifier gradient. As the formulation oils were unretained, good separation of the oil matrix and the API was achieved, with no impurity co-elution issues and with simpler sample preparation than the RPLC method.

Figure 1a and 1b shows the overlay of the chromatograms of API (black trace) dissolved in dichloromethane (DCM, red trace) versus the formulation matrix oils (blue trace). Acetone was later selected as the method diluent to replace DCM as a “greener” option (5).

Figure 1: Overlay of the SFC chromatograms of an API (black trace), its formulation excipients (blue trace), the sample diluent (DCM) – blank (red trace). (a) full scale, and (b) zoom (Torus DIOL (100 x 3.0 mm, 1.7 µm), methanol 10 mM ammonium formate (2%, hold 0.5 min, to 42% in 10 min), 40 °C, 120 bar, 1.7 mL/min, 2 µL, 292 nm).

The drug product application of SFC detailed above started with a risk assessment of the sample characteristics versus the ATP. It is an example of a science-based approach to successful method development. The method development took no more than one day, compared to several weeks dedicated to RPLC method development and troubleshooting. This highlights the alternative utility of SFC in pharmaceutical analytical laboratories with challenging matrices.

SFC in the QC Laboratory: API Manufacture and Release Testing

The superiority of SFC for chiral separations is well known and widely described in the literature (6–9). SFC has been the technique of choice for decades in compound discovery departments and for preparative and purification separations. It is only recently that SFC technology became reliable enough that its use could satisfy the strict requirements for validation, method transfer, and ultimately implementation in the QC labs. Several reports have been presented in the literature highlighting GMP applications in recent years (10–13).

Implementing modern SFC instrumentation in QC laboratories has added a new platform for the chiral control strategy of APIs which were predominantly being analyzed using normal-phase LC (NPLC). While the use of NPLC for chiral measurements can be satisfactory, due to long analysis timesand toxic or non-green mobile phases incorporating organic solvents such as hexane or heptane, it is rarely the optimum separation approach.

Figure 2 displays the increase in number of batches tested with SFC as part of Pfizer’s API clinical manufacturing control strategy (Sandwich, UK R&D site) from 2016 to 2023. As can be seen, SFC has replaced NPLC for clinical API manufacturing support almost entirely over this timespan.

Figure 2: SFC vs. NPLC in Pfizer, GMP Analytics (Sandwich) for release of Clinical API, Intermediats and IPCs. Bar graph shows the number of batches tested at Pfizer’s API clinical manufacturing (Sandwich, UK R&D) site from 2016 to 2023 by SFC (blue bars) and by NPLC (orange bars).

The confidence in the robustness of SFC methods created during development, allowed Pfizer to transfer SFC methods to commercial operations. Between 2016 and 2020, SFC chiral methods were redeveloped to NPLC equivalents at point of registration or commercial transfer due to lack of SFC systems in manufacturing units. Since then, three marketed products have been registered with SFC methods as part of their control strategy:

• Abrocitinib (cibinqo): Determinaiton of process impurity content in water-sensitive intermediate by SFC where NPLC gave poor and unreproducible chromatography.
• Ritlecitinib (litfulo): API identity and chiral purity evaluation by SFC.
• Nirmatrelvir (paxlovid): Determination of API stereoisomer content by SFC.

The transfer of abrocitinib in 2020 from clinical to commercial manufacture opened the door for SFC to Pfizer’s commercial manufacturing partners.

Two subsequent commercial chiral API methods were successfully transferred to Pfizer commercial API sites, without method transfer issues. Furthermore, the SFC methods were well received by the QC teams.

Since its implementation in the Ringaskiddy (Ireland) API manufacturing site, over 110 individual release runs were completed using SFC since 2020, over half of which were for Nirmatrelvir. Modern SFC instrumentation has proven to be very reliable in this environment.

Nitrosamine Determination

In 2020, the FDA published guidance (14) discussing the risk of nitrosamine drug substance related impurities (NDSRIs) and mitigation strategies. In 2023, the FDA published recommended acceptable intake limits for these mutagenic impurities (15). While a risk assessment and mitigation approach can be taken for the control of NDSRIs, identification and quantitation by analytical methods are still required to appropriately support the risk assessment. Here, we consider SFC as a technique to aid in the control strategy of NDSRIs (16).

Sensitivity to low- or sub-ppm levels are required to reliably determine levels of NDSRIs, often requiring mass spectrometric (MS) and tandem mass spectrometric (MS/MS) detection. While nitrosamines are often analyzed by GC or RPLC, SFC can also be considered for the following reasons:

1. Orthogonal selectivity to RPLC: Nitrosamine derivative of the API often are eluted after the API with RPLC, whereas they often are eluted prior to the API using SFC which can, in turn, aid quantification.
2. Compatibility of SFC with MS detection.
3. Often enhanced MS sensitivity due to simpler mobile phase desolvation.
4. High on column drug loading: High concentration samples are possible through dissolution in organic solvents, such as acetone. These strong organic strength solutions are highly compatible with SFC.

Figure 3 shows the total ion chromatogram (TIC) plots of an (a) SFC–MS chromatogram, and (b) RPLC–MS chromatogram. The nitrosamine derivative is eluted before the main component in SFC–MS providing a reduced risk of matrix effects in the ion source and improved quantification.

Figure 3: Total Ion Chromatograms (TIC) – API (30 mg/mL) spiked with NO-Am (0.05 mg/mL) for (a) an SFC–MS (Torus DIOL (100 x 3.0 mm, 1.7 µm), methanol 10 mM ammonium formate [5% to 20% in 20 min], 40 °C, 120 bar, 2.0 mL/min, 10 µL); and (b) LC–MS, RPLC (Zorbax Eclipse Plus [100 x 2.1 mm, 1.8 μm], MPA: 10 mM ammonium acetate, pH 4.5, MPB: acetonitrile, gradient 20% hold 2 min to 80% in 13 min, 0.3 mL/min, 40 °C, 10 µL injection vol.) of an API spiked with its related nitrosamine.

Conclusion

Systematic science-based method development is essential to ensuring analytical methods can successfully support drug development through the entire lifecycle. We have shared a series of applications demonstrating successful method development and validation with SFC. We have demonstrated the application of SFC in QC and commercial manufacturing support and highlight how the technique can be used beyond discovery applications. Additionally, through these examples, we have shown SFC can provide a higher degree of speed, robustness, and simplicity over alternative or more established techniques.

Acknowledgments

We thank Liliana Silva for the evaluation of the SFC–MS for the nitrosamine determination.

References

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About the Authors

Claudio Brunelli and Wayne Callar were with the Analytical R&D department of Pfizer UK R&D Ltd, in Kent, United Kingdom at the time of the submission of this article. Direct correpondence to: Claudio.Brunelli@gmail.com