Development of Stability-Indicating Analytical Procedures by HPLC: An Overview and Best Practices

This installment is the second in a series of three white papers on stability studies and testing of pharmaceuticals. It focuses on the development of stability-indicating high performance liquid chromatography (HPLC) methods for drug substances and products. This paper provides an overview of the fundamentals and best practices in method development, including the traditional and other easier approaches, as well as modern trends and software tools for expediting the process. The regulatory guidance on the necessary contents of a well-written method, strategies for efficient method execution, and life cycle management of analytical procedures, are described.

High performance liquid chromatography (HPLC) method development can be a time-consuming process, particularly for stability-indicating analytical procedures of new chemical entities (NCEs). Most of the procedures for small-molecule drugs employ gradient reversed-phase liquid chromatography (RPLC) with ultraviolet (UV) detection. These methods are designed to separate and quantitate the active pharmaceutical ingredients (APIs) and all process impurities and degradation products in drug substance (DS) and drug product (DP) samples. This important HPLC method category provides quality assessment data required in product release and stability studies in regulatory filings and procedures (Table I). Information on the HPLC method development process and requirements are available in many textbooks (1–6), journal articles (7–13), regulatory guidance documents (14–16), and other resources (17). In this installment, we strive to provide a concise but comprehensive overview of the essential steps of the method development process, supported with case studies, common practices, regulatory guidance, and citations of critical references.

Why Gradient RPLC with UV Detection for Stability-Indicating Methods?

Let us start with the fundamentals and the rationale for the use of RPLC and UV methods in stability-indicating analytical procedures, which must separate the API from all impurities, and meet stringent regulatory requirements in method performance (6,14–16).

First, the primary retention mechanism in RPLC is hydrophobic interaction, particularly useful for compounds with intermediate polarities, and an excellent match of most small-molecule drugs with oral bioavailability (2). The weak dispersive forces in RPLC ensure that all components in the sample can be eluted from the column using strong purging solvents at the end of the gradient, thus yielding 100% recovery of the injected sample.

Second, the elution order in RPLC is highly predictable. It follows the “Linear Solvent Strength Model (4).” In most cases, the log k (retention factor) of the analyte is inversely proportional to %MPB (mobile phase B or % of the strong organic modifier). In addition, most drugs are basic, but have sufficient hydrophobicity to be retained in RPLC in the ionized forms in acidic mobile phases. The retention of the “solvated” analytes can be manipulated to provide increased resolution by changing the composition of the mobile phase (for example, water, organic solvents, and pH), that interacts with the analytes in the “solvated” state, and moderates their retention on the stationary phase (1,2). Gradient elution is commonly used in stability-indicating assays to yield higher peak capacity and sensitivity for both hydrophilic and hydrophobic components in the sample (2).

Third, most NCEs are chromophoric, having one or more conjugated double bonds or aromatic moieties in their structures, thus providing high detection sensitivity with UV detectors. A peak area precision of 0.1–0.5% relative standard deviation (RSD) is routinely achievable in HPLC-UV assays, because the entire injected sample passes through the UV flow cell. This high precision performance is necessary for quality control applications for DS release testing (with typical specifications of 98.0–102.0%). In contrast, the precision performance of <1% is difficult to achieve with mass spectrometry (MS) detection, because only a small fraction of the nebulized eluent stream is ionized and detected (2). Furthermore, a UV detector offers a wide linear response range exceeding five orders of magnitude (1 x 10-5 to ~2 absorbance units [AU]), allowing the use of a single-point calibration curve for early-phase assays, and normalized area % analysis for the determination of impurities (2).

There are some notable exceptions to the adoption of RPLC-UV methods. For NCEs with no or low chromophoric properties, a gradient-compatible near-universal detector, such as a charged aerosol detector (CAD) or an evaporative light-scattering detector (ELSD), is typically employed (2). For the determination of enantiomers, a normal-phase separation using chiral LC or supercritical fluid chromatography (SFC) separation, is generally preferred, because the analytes are present in an unassociated and non-ionized state, which often leads to higher selectivity differences between the enantiomers (2).

Another exception is the determination of potentially genotoxic impurities at parts-per-million levels (not discussed here), which may require a high-sensitivity analytical procedure, such as
LC with mass spectrometry detection (LC–MS) (2).

Objectives of This Article

The objectives of this installment are as follows.

Describe the traditional five-step strategy in HPLC method development, according to Snyder and associates (1), and illustrate the use of the selectivity-tuning for method optimization with case studies.

Describe modern HPLC method development trends using ultrahigh-pressure liquid chromatography (UHPLC), MS, automated column or mobile-phase screening systems, and software platform tools.

Introduce easier or more straight-forward method development approaches, including a three-pronged template for early-phase development and universal generic gradient methods.

Describe the essential elements of a well-written analytical method, strategies for effective method execution,and the concept of life cycle management of analytical procedures.

The Traditional Five-Step Method Development Approach

A traditional five-step approach for HPLC method development was proposed by Snyder and associates, based on the concept of selectivity-tuning to increase the resolution of critical pairs (close-eluting peaks) for accurate quantitation (1,2). The five-step approach is outlined here.

Step 1: Defining Method Types

The first step is to define the method type. Is it a preparative, qualitative, or quantitative method? The most common method type is a stability-indicating analytical procedure (also known as an assay and impurities determination) for the quantitation of the API and impurities in pharmaceuticals. It is a challenging method development task to develop this type of method, because all key components in the sample must be physically separated in one chromatogram with method performance compliant to ICH guidelines (14). In comparison, other pharmaceutical methods for identification, limit tests, or performance assays, are simpler to develop, validate, and execute. Typically, the DS method is developed first for the NCE, and the DP method is then optimized based on the DS method.

Step 2: Gathering Sample and Analyte Information

The next step is to gather information on the sample and analytes. For NCEs, the structures and molecular formulae are well established, allowing the inference or calculation of physicochemical properties, such as pKa, logP, logD, polarity, numbers of acidic, basic, or aromatic functional groups, and chiral centers. These characteristics can be useful in the selection of columns, mobile phases, and sample diluents. The pKa values can be used to select a mobile- phase pH that ensures the compound is in a singly charged state. Knowledge of the acidic, basic, and aromatic functional groups can be helpful in column and mobile-phase selection, and provides forewarnings of potential stability or reactivity issues. Finally, knowing the presence and number of chiral centers is vital for planning an analytical procedure that includes diastereomeric separations. Though not entirely in the scope of this article, possible diastereomeric combinations may be separated with an achiral RPLC column (shown in the case study in Figure 5). Enantiomeric separation offers a different challenge that will require selecting the appropriate chiral-selective column for the determination of enantiomers of the API using a different analytical procedure. To gather analyte information, resources such as Certificate of Analysis (CoA) from suppliers or technical packages from the API manufacturers are invaluable in obtaining initial information regarding sample purity, methodologies, spectral and safety data, and other attributes of the API.

Step 3: Initial Method Development

This is the first laboratory-based step in the development process, and it involves performing “scouting” runs to obtain the first chromatograms. Details in column and mobile-phase selection are covered in many books (1–3). To illustrate the initial method development step, we will start here with the most common choice of a C18 column used with an acidified aqueous mobile phase and organic solvent. Step #3, outlined here, is extracted from a case study published in this reference (2).

Here, a sample of the API is dissolved in a default diluent (in this case, 1 mg/mL in 50% acetonitrile in water) and injected into an HPLC-UV system (with a photodiode array (PDA) detector and an MS instrument. A common broad-gradient RPLC method can be used (such as mobile phase A [MPA] = 0.1% formic acid in water; mobile phase B [MPB] = acetonitrile; C18 column: 3-µm, 100 mm x 3.0 mm i.d., 5–100% acetonitrile in 10 min at 1 mL/min and a column temperature at 30 oC). Full spectral data in UV (220–400 nm) and MS (100–1200 amu with positive ionization) are collected to allow reconstruction of chromatograms at any monitoring wavelengths.

It is important to choose an MS-friendly mobile phase in this step if possible, to reduce the need for any method changes when using MS later. Results from the first scouting run can generate pertinent data, such as a “rough” sample impurity profile, estimation of purity and hydrophobicity of the API, maximum absorbance wavelength (λmax), (M+H)1+ data of the NCE, and any observed impurities. These initial data are used for determining the next logical steps in method fine-tuning.

The process is often modified for polar or water-soluble NCEs, which may be better retained on an AQ-type-C18 or polar-embedded column (2). For drug candidates with low or no UV chromophoric activities, detectors such as CAD, ELSD, or MS may be employed. One crucial issue in the initial method development of NCEs is the rare availability of an “ideal test mix” sample containing the API and all key impurities or degradation products, deterring a systematic application of automation systems. This dilemma of sample availability will be discussed later.

Step 4: Method Fine-Tuning and Optimization

This is the most time-consuming step for the development of stability-indicating procedures. The process is typically reliant on “selectivity tuning” by changing selectivity (α) in a rational fashion by adjusting mobile phases (organic modifier, pH, buffer strength) and operating parameters (flow [F], gradient time [tG], column temperature [T]) (1–3). Often, changing to a column packed with different bonded phases other than C18 may be needed (such as, for example, phenyl, polar-embedded, or cyano) to achieve the separation of a critical pair of coeluting peaks.

The next step is often to employ a “shallow middle gradient segment” indexed to the hydrophobicity of the API to increase the resolution around the main component (2). This use of a “multi-segment gradient approach” is preferred for complex NCEs and is discussed later.

The method fine-tuning or optimization process typically takes a few days or 1 to 2 weeks, depending on the complexity of the NCE and the availability of key impurities as reference materials (such as isomers) that are used as retention time markers. This process is often iterative, and is performed before or after the initial forced degradation studies where degradation products are generated to challenge the separation power of the initial method. Peak purity should be evaluated with PDA and MS. Quite often, the initial column used (for example, C18) is found to be inadequate in the separation of a critical pair (for example, API and the immediate synthetic precursor) (2). This would invariably trigger a column screening experiment to find a better column with a different selectivity (for example, phenyl, polar-embedded, or cyano) (2,3). The use of MS can be a valuable tool in column screening, as it can help to track known impurities from column to column as well as degradation products formed during forced degradation studies. Knowledge of the molecular weights helps to determine the molecular structures of the degradation products.

For laboratories specializing in method development, the use of automated column and mobile-phase screening systems and other software platforms can expedite this time-consuming step. The last steps in the method development phase are the final minor method adjustments needed to improve sensitivity and peak shape (injection volume, sample concentration, diluent, monitoring wavelength), and analysis time.

It should also be noted that the drug product will also require assays and impurity analysis throughout the life cycle of the NCE. The best and shortest approach to DP method development is to use the chromatographic conditions for the DS with a modified sample preparation procedure.

Step 5: Method Prequalification

Step 5 for methods used to test regulated products is a method prequalification or prevalidation step. This step will ensure the method can be successfully validated by determining the potential to pass typical method validation criteria (2,16), including specificity, precision, linearity, sensitivity, and often accuracy also. This prequalification step can take from a few hours to 1 or 2 d for most methods.

Examples of the Selectivity Tuning Approach by Changing Mobile Phase, Column, or Both

Figures 1 to 3 show studies of the use of selectivity tuning by changing the mobile phase, column, or both. Figure 1 shows the separation of a 12-component test mixture of basic, acidic, and neutral drugs and the changes in peak spacings with the gradient separation on a C18 column by switching the organic mobile phase (MPB) from methanol to acetonitrile. Note that acetonitrile is a stronger organic modifier in RPLC; thus, the elution time is considerably shorter. The peak shapes are also sharper due to its lower viscosity (1,2). While the elution order remains the same without any peak crossovers, the band spacings are changed due to selectivity differences. Note that acetonitrile is an aprotic solvent, while methanol is capable of hydrogen bonding and polar interaction with the analytes. In cases of analytes that can hydrogen-bond or have polar interaction, the elution order may be different when changing from methanol to acetonitrile.

Figure 1: The comparative chromatograms illustrate the use of selectivity tuning by different organic solvents. Chromatograms courtesy of Waters Corporation.

Figure 2 shows eight comparative chromatograms of a 7-component mixture of basic drugs using columns packed with different types of C18, phenyl, cyano, and pentafluorophenyl phases from a single manufacturer. All columns have the same dimension and are packed with similar particle sizes of ~1.7 µm. Chromatograms show similar elution order but with many differences in band spacings for C18 phases, whose predominant retention mechanism is hydrophobic interaction. However, the elution order can be substantially different in columns packed with different bonded phases that have additional retention mechanisms from π-π, polar, and hydrogen bonding interactions. To find the best column for a specific separation, the most efficient approach is to use an automated column and mobile phase screening system (2,3,12).

Figure 2: Comparative chromatograms of the gradient separations of a test mix containing seven basic drugs, illustrating the effect of selectivity of bonded phases (C18, phenyl, cyano, pentafluorophenyl [PFP]). All columns are 50 x 2.1 mm, 1.7-µm, with 10-mM ammonium formate at pH 3, and the same linear gradient with methanol. Diagram courtesy of Waters Corporation.

Figure 3 shows two comparative chromatograms in a case study on proactive phase-appropriate method development advocated by Rasmussen and associates (3,17). The top chromatogram shows the separation of a retention marker solution of an API spiked with available impurities that is analyzed by a primary stability-indicating method for a DS method using a C18 column and an MPA at pH 2.5. The bottom chromatogram shows the separation of the same test mix using the secondary orthogonal method on a polar-embedded phase at a neutral MPA pH. More discussion on the development of a secondary orthogonal method is found in a later section.

Figure 3: Two comparative HPLC chromatograms of a primary regulatory method (top) and a secondary orthogonal method (bottom) used in the phase-appropriate method development approach advocated by H. Rasmussen and associates (3). Reprint from reference (3).

A Summary of Suggested Parameter Selections for Steps 3 and 4

A summary of suggested parameter selections for steps 3 and 4 is listed in Table II as a reference guide for default conditions in HPLC method development for NCEs.

During the life cycle of any DS or DP, the analytical methods may need to be redeveloped. A new synthetic route or process chemistry changes during scale-up may cause new impurities to be present in the final DS. Also, degradation products found in samples from stability studies may require an improved resolution for quantitation.

Method Development Approaches and Automation Tools

In this section, we describe modern approaches in method development and common automated tools for expediting the process. The reader is referred to textbooks, articles, and manufacturers’ websites for updates and more complete descriptions (18–29). Prior to discussing the modern development approaches, let us discuss briefly sample diluent selection for the NCE and sample preparation for DS and DP.

Selection of Sample Diluent and Sample Preparation Procedure

Knowledge about the solubility properties of the API is necessary to choose the most appropriate sample diluent. The most natural choice is to use the initial gradient concentration of the mobile phase. Although this approach may work for hydrophilic or water-soluble compounds, many hydrophobic compounds may not be fully dissolved in these mostly aqueous diluents. Increasing the organic phase concentration may result in full dissolution. However, a higher concentration of organic solvent in the sample diluents may cause chromatographic peak anomaly issues, such as peak splitting upon the injection of large sample volumes (2). Another approach may be the use of a stronger solubilizing agent (such as DMSO), in small concentrations to solubilize the compound and then dilute with the starting concentration of the mobile phase. Often, vigorous shaking manually or with a shaker, or sonication, may be required to expedite the solubilization process. Filtration of a drug substance sample is not recommended since the material must be completely dissolved in a solution for accurate quantitation.

Drug products, such as tablets, also have the added complications of the need to break down the formulation matrix to permit efficient and complete extraction and solubilization of the active ingredients. The separation can be especially problematic upon stability storage due to the aging of the dosage form. Multi-step dilution schemes using shakers or sonication in an appropriate extraction medium followed by filtration (such as 13–25 mm, i.d., 0.2–0.45-µm disposable membrane syringe filters) or centrifugation is generally required to obtain the final sample DP extract for analysis. For sustained-release drug product formulations, a 2-step extraction process is often needed, using an organic solvent to dissolve the polymer-based matrix in the first step. Grinding or rough-crushing the formulation may also be needed to expedite the extraction process. More detailed procedures are available in these references (2,3).

Modern Method Development Approaches and Trends

UHPLC

UHPLC is a modern low-dispersion system platform with higher-pressure ratings (15,000–22,500 psi), and is the most significant innovation in HPLC in recent years (18–23). The benefits of UHPLC, when coupled with small-particle columns (sub-2 and sub-3-μm), are faster analysis, enhanced sensitivity, and higher peak capacities for sample analysis (13, 22). The lower system dwell volumes and faster column equilibration times for the smaller UHPLC columns provide another significant benefit for the method development process (12,22). In method optimization, the mobile phase and operating conditions are varied in numerous trial experiments and iterations to optimize the chromatographic behavior of all key analytes to arrive at the optimal separation. The judicious application of UHPLC can reduce method development times by a factor of 3 to 5 in comparison with those obtained with conventional HPLC systems and columns. In addition, UHPLC significantly reduces the amount of solvents used and solvent waste (22).

A potential issue for using UHPLC in method development is the necessity to “back-translate” (also often called “back-transfer” in the literature) or convert the UHPLC methods back to HPLC conditions because many QC laboratories may not be equipped with UHPLC (2,18,23). Today, 16 years after the commercialization of the UHPLC system in 2004 (18), the need for changing the method from UHPLC back to HPLC is lessened by the wider availability of these UHPLC systems (>15,000 psi), and others with intermediary pressures (for example, 9,000–2,000 psi) (19,20).

MS

While most stability-indicating analytical procedures developed for NCEs are HPLC-UV methods, many are also beneficially designed to be MS-compatible using volatile reagents in the mobile phases to allow peak verification, tracking, and identification (2,3). The inclusion of an information-rich MS in the HPLC-UV method development system, in our view, is mandatory for a science-based and efficient method development process, and particularly useful for developing methods of complex NCEs. The MS detector is immensely helpful in the case that the impurities must be identified or qualified.

The advent of low-cost, easy-to-use single-quadrupole MS (SQMS) makes this adoption much easier for any analytical chemist without specialized MS training (2,19). The only downside is the loss in sensitivity and linear performance for detection wavelength <230 nm due tothe end absorbance of the volatile mobile phase additives (such as formic acid).

Phase-Appropriate Method Development and Validation

The concept of “phase-appropriate method development and validation,”’ advocated by H. Rasmussen and associates and others in the early 2000s, recognizes that it is unrealistic to expect the original stability-indicating method for an NCE to remain unchanged during the life cycle of a new drug candidate to commercialization (3,17). It is, therefore, more realistic to have a phase-appropriate or tiered approach, in which the initial method is continuously improved as the formulation is being developed. Complete validation studies are conducted as the project is advancing to late-phase development when the synthetic chemistry process and drug product formulation are finalized.

The approach recommends the development of a secondary orthogonal method early for several advantages (17). While the word orthogonal separationgenerally means using different retention mechanisms (for example, RPLC based on hydrophobic interaction vs. capillary electrophoresis based on the ionic mobility of the analytes), in reality, most orthogonal methods employ two columns with different selectivity (C18 vs. polar-embedded) or the same column using different organic modifiers (acetonitrile vs. methanol). The secondary orthogonal method ensures that there is no hidden impurities underneath the API peak. It can be used to analyze pivotal clinical lots for supporting data. A minimum method validation can be done to expedite the testing. In addition, the orthogonal method can be used when the primary method is found to be inadequate (such as the inability to resolve a new impurity). This approach can reduce the risk of a potential method deficiency situation, which can have a severe impact on the drug development timeline. Case studies to illustrate this approach are shown in Figures 3 and 5.

Applications of Quality by Design (QbD) and Design of Experiments (DoE) in Method Development

Regulatory authorities have endorsed a science- and risk-based strategy in new drug development, using a systematic Quality by Design (QbD) approach in process development and control of critical raw materials (24). The QbD concept can also be used in method development to establish the capabilities of the method. While a traditional “one-factor-at-a-time”approach is widely practiced in method development, many laboratories have started to use a Design of Experiments (DoE) approach for method robustness validation (3,5). Others have explored the use of automated scouting or screening systems and software platforms for the method development process (2,3,25–27).

Automation and Software Platform Systems for Method Development

Although most HPLC systems can be post-fitted with selection valves to automate the column/mobile phase screening studies (2,3,12), many manufacturers now offer preconfigured method scouting and screening systems, often with some simple optimization algorithms. They can also work with more elaborate software platforms to facilitate the method development process (2).

Table III lists automated method scouting or screening systems and software platforms for HPLC method development. Many software platforms are available for off-line predictive modeling with inputs from limited experiments (such as DryLab) (2,4,25). Other platforms support direct instrument control and on-line optimization (such as ChromSword Auto and ACD Labs/AutoChrom) and statistical analysis of experimental results (such as Fusion QbD) (2,26–27). The reader is referred to published journal articles and the manufacturers’ websites for further details (25–27).

A Dilemma in Early-Phase Method Development

The use of automated and software systems can expedite the method development process, particularly in laboratories specializing in late-phase product development situations where critical process impurities and degradation products are identified and available as reference materials. This ideal test mix containing all the critical impurities is rarely available during initial method development for an NCE, making it impossible to have a systematic application of automated workflows in early development.

Nevertheless, the analytical chemist still needs to have a reasonable first HPLC method to initiate sample evaluation from forced degradation studies. The most realistic scenario is to have proactive communication with the process chemist and to obtain the mother liquors of the final crystallization of the API. As a minimum, a mixture of precursors, starting materials, intermediates from previous synthetic steps, plus any additional reference materials available such as stereoisomers, can be assembled for use as method development samples (3,17).

Easier Approaches to MethodDevelopment for Pharmaceutical Analysis

The traditional five-step approach with judicious choice of column, detector, and mobile-phase conditions works well for systematic method development for pharmaceutical analysis when implemented by experienced scientists. However, it would be desirable to have easier approaches to develop effective methods by analysts with diverse experience levels. Here we will describe two such approaches.

A Three-Pronged Template Approach

A three-pronged template approach was proposed in 2013 to facilitate the rapid development of early-phase RPLC methods by recognizing the attributes of the three types of HPLC methods commonly used in the pharmaceutical analysis (10). These method templates, with their unique sets of characteristics and capabilities, allow a quicker method development process for scientists by recognizing the attributes and the expected final method conditions (column, operating conditions). These method templates are described below.

Template 1. Fast (sub-2-min) LC isocratic methods for rough assays (potency of DS or strength of DP) used in content uniformity and dissolution (performance) testing of drug products (2). These are non-stability-indicating procedures for the determination of the main component (assay) only. They can be developed and qualified quickly. Note that this method type is not adequate to be used for actual potency assay of a DS or purity factor of a DS on a certificate of analysis (CoA), which must be derived from a validated stability-indicating method or method template #3.

Template 2. Generic broad-gradient RPLC methods can be used for high-throughput screening (1–2 min) (5,28) or in-process control testing (4–10 min) (2,5). Still, the broad gradient RPLC methods can also be re-purposed for purity assays of simpler samples such as raw materials, starting materials, reagents, and cleaning verification (30) samples. The generic methods can be easily modified for stability-indicating assays of simpler drug molecules by extending the gradient times (tG) or using a more complex gradient profile. An organization can adopt a few generic broad-gradient methods for applications across different early development projects to reduce to burden for method development. This practice can save considerable analytical resources, reduce the method development, validation, and transfer time, and the stockpile of different HPLC columns by individual scientists within an organization (29).

Template 3. Multi-segment gradient methods. A brief survey of literature conducted in 2013 indicated a predominance of multi-segment gradient methods for ICH-compliant stability-indicating analytical procedures for complex NCEs (2,10). The rationales for this unique gradient pattern (see Figure 4) are likely to be as follows:

Figure 4: An example of a gradient profile of the “multi-segment gradient method template #3” using a shallow middle segment (15–40% B in 25 min) with the main component eluted toward the end of the gradient segment. This segment is preceded by an early segment (5–15% B in 5 min) and followed by a steep segment (40–90% B in 3 min) with a 2-min purging step at 90% B. Reprinted from reference (10).

Isomers, impurities, and degradants of the API usually have similar structures and hydrophobicity to those of the parent molecule and thus may be eluted close to the API under RPLC.

A shallow gradient with the API eluted toward the end of the gradient segment would maximize the resolution around the API peak. The hydrophobicity of the API defines the final %MPB of this segment.

A steep gradient segment, followed by a purging step, is used to elute highly-retained components (dimers). An initial low-strength gradient segment (starting at 2–5% MPB) may be required to retain polar impurities (such as hydrolytic degradants).

Traditionally, a single, broad, linear gradient is used during initial method development and column screening. This fast gradient approach is acceptable for initial phase screening but is unlikely to provide sufficient resolution around the API to resolve closely eluted impurities. The understanding of the rationales for the multi-segment gradient program offers helpful insights into the fine-tuning method development steps used in method optimization (step 4). Note that linear gradient segments are invariably used, and isocratic steps should be avoided to reduce method transfer issues between different HPLC or UHPLC systems.

Case Studies to Illustrate the Use of Multi-Segment Gradient Approach for Complex DS and DP Methods

Two case studies are included here to illustrate the application of this multi-segment gradient approach in the method development of complex pharmaceuticals (13). The third case study is an early UHPLC method development example of an NCE DS method and the “back-transfer” to HPLC conditions (9).

A Complex NCE with Three Chiral Centers

Figure 5a shows a chromatogram in a method development case study for a complex molecule with three chiral centers. Here, the resolution of the four pairs of diastereomers becomes the crucial criterion for quality assessment (13). The middle gradient segment employed is a shallow gradient (15–40%B in 25 min) used to separate the API (with an absolute configuration of SRR) from its diastereomers (SRS, RRR, and RRS) and other impurities (M416, M456, and M399, designated as their parent (M+H)1+ ions). A low-strength initial gradient allows for the retention of a hydrolytic degradant (M235), while the final segment depicts a steep gradient that elutes any dimers or other late-eluting species. Note that this 42-min HPLC method has been converted into a 17-min UHPLC method using a 100-mm column packed with 2-µm particles with equal resolution performance (13).

Figure 5: Chromatograms illustrating the applications of template #3 in the separation of the four diastereomers in a multi-chiral NCEs with three chiral centers described in reference 13. Column and full operating conditions are shown in the inset. (a) the 42-min primary regulatory HPLC method using a C18 column with acetonitrile; (b) a secondary orthogonal method using a phenyl column and methanol.

Figure 5b shows a chromatogram of the secondary orthogonal method developed at the same time, which yields a better resolution of the diastereomers using a phenyl column and methanol. Nevertheless, the HPLC conditions used in Figure 5a with an analysis time of 42 min was employed as the primary regulatory method due to its higher overall resolution and peak capacity. The case study also illustrates the implementation of a proactive method development approach using a secondary orthogonal method to ensure there are no impurities hidden underneath the API peak (2,3).

A Method for a Complex DP with Multiple APIs

Figure 6 shows a UHPLC-UV chromatogram of a retention marker solution consisting of 60+ components used in a stability-indicating method for a complex DP with four APIs (Strilbild tablet for HIV indication). The method uses a 5-segment linear gradient profile customized to maximize the resolution of the four APIs. The method development process utilized a software-assisted approach using the QbD principle (13,27). This procedure has been successfully validated, and a validation summary data is available from the reference paper (13).

Figure 6: Chromatogram showing a stability-indicating UHPLC method for a drug product Stribild oral tablet with four APIs (elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate). UHPLC method conditions are shown in the inset. Chromatogram reprinted from reference (13).

An Early UHPLC Method Development Case Study of an NCE DS Procedure

Figures 7a–d shows a sequence of four chromatograms obtained during an early UHPLC method development case study of an NCE DS method, which has several light degradants published in 2007 (9). Figure 7a shows the original 35-min HPLC chromatogram from a client using a phosphate buffer. This mostly isocratic method was not acceptable because a shoulder “hump” was observed on the downslope of the API peak, indicating the presence of a hidden impurity. Figure 7b shows a 5-min broad-gradient column screening study using a 50-mm C18 column showing an equivalent separation of the original HPLC chromatogram. The entire 4-column screens study took 1 h, indicating a polar-embedded phase (Acquity Shield RP18, 50 x 2.1 mm, 1.7-µm) yielded the best separation around the critical region. MPA was changed to an MS-compatible buffer of 20-mM ammonium format at pH 3.7. Figure 7c shows the final 13-min UHPLC method using an Acquity Shield RP18, 100 x 2.1 mm, 1.7-µm, which yielded an improved resolution of three impurities peaks in the critical region. Figure 7d shows the final 20-min “back-translated” HPLC method using an HPLC column (XBridge Shield RP18, 150 x 3.0 mm, 3.5-µm). This process took 2 d, using principles of “geometric-scaling” (23), assisted by a modeling software (DryLab) to ensure that all three light degradants labeled with an asterisk could be resolved. The entire method development project took about 1 week, including the “back-translation” of the UHPLC method into HPLC conditions.

Figure 7: A case study of the UHPLC method development of a DS method of an NCE with several light degradants. (a) The original 35-min HPLC method using a phosphate buffer at pH 3.1; (b) A 5-min broad-gradient column screening study using a short C18 column; (c) The final 13-min UHPLC method (Acquity Shield RP18, 100 x 2.1 mm, 1.7-μm) yields three impurities peaks in the critical region; (d) The final 20-min “back-translated” HPLC method using an HPLC column (XBridge Shield RP18, 150 x 3.0 mm, 3.5-μm) showing the resolution of three light degradants.

A Modernized Universal Generic Method for Multiple NCEs

Wouldn’t it be nice if we could have a single universal generic gradient method that works for multiple NCEs and most pharmaceutical analyses? The concept appeared to work well for cleaning verification when a 10-min HPLC-UV generic method was found suitable for multiple NCEs in a small-molecule portfolio of an organization in 2012 (30). In 2014, the idea was further extended by applying the latest column technologies (short columns packed with sub-3-µm superficially porous particles (SPP) with improved bonded chemistry) to allow the development of a modernized version of a 2-min generic gradient method operating at optimum conditions with simple mobile phases (11).

Figure 8a shows the performance of this modernized generic gradient HPLC-UV–MS method in the separation of a 12-component test mix (11). The method has a peak capacity (Pc) of 100, and resolves ten peaks out a mixture of 12 NCEs with good peak shapes and sensitivity with simple mobile phases on both HPLC and UHPLC systems (3700 psi). Best of all, the “standard” 2-min method can be easily adjusted to resolve all twelve NCEs with a run time of 5 min by simple adjustments of gradient conditions (Figure 8b).

Figure 8: (a) The separation of a 12-NCE mixture using the proposed 2-min universal gradient reversed-phase chromatography method using a sub-3-μm superficially pourous particle column. (b) A chromatogram depicting the achievement of near-baseline resolution of all 12 NCEs in the test mixture by adjustments of gradient conditions using the same bonded phase in a 2.1 mm column format. Each peak is designated by its code name, retention time in minutes, and (M+H) +1 parent ion. Reprinted from reference 11.

The rationales in the selection of columns, mobile phases, and operating conditions are explained in the 2016 reference (11). This reference also provides several case studies that this generic method can be applied to stability-indicating applications by quick adjustments of gradient conditions (using longer tG and multi-segment gradient) to obtain reasonable initial method conditions. The next steps can be a column or mobile-phase screening and the use of a longer column to arrive at the final regulatory method conditions.

In summary, a generic gradient method using a short column operating at optimum conditions shown in Figure 8a (for example, C18, 50 mmx 3.0 mm i.d., 2.7-µm SPP, 5 - 60% acetonitrile in 2 min at 1 mL/min) is capable of delivering peak capacities (Pc) of 100-300 in 2 to 10 minutes (2,31–32). The generic method approach works well for cleaning verification of multiple NCEs and provides a useful starting point in the development of stability-indicating analytical procedures.

Method Content, Execution, and Life Cycle Management

We devote the final section to discussing the essential elements in a well-written HPLC method (33), guidance in method execution to generate more reliable stability data, and the concept of life cycle management of analytical procedures (34).

Method Content and Essential Elements in an Analytical Procedure

A well-written analytical procedure with all essential elements is critical for its successful execution of the laboratory. Table IV lists these essential elements for late-stage methods, excerpted from a recent U.S. FDA guidance document (33).

In addition, it is also essential to include as many helpful details in the written method as the analyst will need to successfully execute the method. The method should include example chromatograms (both full- and expanded-scale of a blank, retention marker solution or SSTsolution, standard solution, and a typical sample solution), safety precautions, a table of RRT and RRF if available (2), and method history with reasons for revisions.

Strategies for Method Execution to Generate Reliable Stability Data

A well-developed and written analytical procedure with full validation and vigorous transfer process is the cornerstone for effective method execution. Understanding the chemistry of the NCE, the physicochemical characteristics of the molecule (such as photosensitivity, hygroscopicity, forms, reactivity, and chirality) and their effects on the stability profile of the DS and DP is crucial. The judicious use of forced degradation and accelerated stability studies, and predictive software models (7) can often reduce the need for conducting more stability studies.

Ideally, the CMC analytical team lead, having intimate knowledge of the drug candidate, should be responsible for the method development and conducting the initial release testing and stability study of the first clinical batches. This work allows a better understanding of the stability profile of the NCE and the nuances in method execution before outsourcing stability studies to external contract organizations. In general, the contract manufacturing organizations (CMOs) selected for the large-scale production of clinical trial materials (CTMs), tend to be preferred partners for conducting stability studies, because they have a higher stake in the success of the development project. It is customary to include shipment of the SST retention marker solutions to the transferred laboratories to lessen the occurrence of peak misidentification caused by retention time peak shifts and potential stability data issues down the line.

Life Cycle Management of Analytical Procedures

The life cycle management approach can benefit a laboratory and the drug developer by improving the performance of methods, reducing analytical uncertainties such as out-of-specification (OOS) and out-of-trend (OOT) results, and improving the success of the method transfers.

Life cycle management of analytical procedures is an integrated method design, development, and performance characterization strategy based on a structured and systematic approach using concepts of QbD similar to those applied to new drug development (24,34). This approach should be phase appropriate during the early drug substance method development to the final fully validated late-stage drug product methodologies. The strategy starts with the definition of an analytical target profile (ATP)–where the requirements of the analytical procedure (what to measure, method and sample type, separation criteria and performance requirements) for assessing the critical quality attributes (CQAs) of the drug substance and the drug products are described. The three stages of the life cycle are:

Stage 1: Method Design and Development

This stage includes an understanding of the variability of the method, risk assessments, and proposed control strategy for critical method parameters (CMPs) that may pose a risk to consistently achieving method requirements.

Stage 2: Method Performance Qualification

The method, with the proposed control strategy, is qualified to ensure it meets its requirements consistently. This stage includes the method validation process and can be phase appropriate depending on the stage of development.

Stage 3: Continued Method Performance Optimization or Verification

This stage includes monitoring the performance after the method is placed into routine QC use by using control samples and charts as well as evaluation and continuous improvements of the method. The results of method transfer can be part of the performance verification process.

Summary and Conclusions

This paper provides an overview of the fundamentals and best practices in the development of stability-indicating methods for pharmaceuticals. It describes the traditional five-step method development approach, as well as modern trends, easier approaches, and software tools for expediting the process. The regulatory guidance on a well-written method, strategy for efficient method execution, and life cycle management of analytical procedures are also discussed.

Acknowledgments

The authors express their gratitude to the following colleagues for their time and efforts to provide timely reviews of the manuscript to improve content accuracy and clarity: Tao Jiang of Mallinckrodt; Anissa Wong and Chris Foti of Gilead Sciences; Mike Shifflet of Johnson and Johnson Consumer Health Care; He Meng of Sanofi; Adrijana Torbovska of Farmahem; Alice Krumenaker of TW Metals, LLC; Mark Shapiro of MCS Pharma Consulting; Szabolcs Fekete of U. Geneva; Deidre Cabooter of KU Leuven; Tamara Andreoli of Medinova AG, Schweiz; Michael DeHart of CMP Pharma; Marc Foster of Mythctest.

References

1. L.R. Snyder, J.J. Kirkland, and J.L. Glajch, Practical HPLC Method Development (John Wiley & Sons, Hoboken, New Jersey, 1997), Chapters 1–2 and 8–9.
2. M.W. Dong, HPLC and UHPLC for Practicing Scientists, (John Wiley & Sons. Hoboken, New Jersey, 2nd Edition, 2019), Chapters 2–6 and 9–11.
3. S. Ahuja and M.W. Dong (Eds.), Handbook of Pharmaceutical Analysis by HPLC (Elsevier. Amsterdam, Netherlands, 2005), Chapters 5 and 6.
4. L.R. Snyder and J.W. Dolan, High-Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model, (Wiley-Interscience, Hoboken, New Jersey, 2007), Chapter 3.
5. Y.V. Kazakevich and R. LoBrutto (Eds.), HPLC for Pharmaceutical Scientists (John Wiley & Sons, Hoboken, New Jersey, 2007), Chapter 8.
6. K. Huynh-Ba, Ed., Handbook of Stability Testing of Pharmaceutical Products: Regulations, Methodologies, and Best Practices (Springer, New York, New York 2009), Chapter 7.
7. M.W. Dong, LCGC North Am. 33(11), 764–775 (2015.)
8. D. Kou, L. Wigman, P. Yehl, and M.W. Dong, LCGC North Am. 33(12), 900–909 (2015).
9. M.W. Dong, LCGC North Am. 25(7), 656–666 (2007).
10. M.W. Dong, LCGC North Am. 31(8), 612–622 (2013).
11. M.W. Dong, LCGC North Am. 34(6), 408–419 (2016)
12. M.W. Dong and K. Zhang, Trends in Anal. Chem, 63, 21–30 (2014).
13. M. Dong, D. Guillarme, D. Prudhomme, et al., LCGC North Am. 32(11), 868–876 (2014).
14. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, ICH Q1A (R2), Stability Testing of New Pharmaceutical Products (Geneva, Switzerland, 2003).
15. International Council for Harmonization (ICH) Q14, Analytical Procedure Development, and Revision of Q2(R1) Analytical Validation (Concept Paper), 2018.
16. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, ICH Q2 (R1), Validation of Analytical Procedures: Methodology (Geneva, Switzerland, 1996, updated 2015).
17. M.W. Dong and H.T. Rasmussen, HPLC Method Development Short Course, Eastern Analytical Symposium, Somerset, New Jersey, 2004.
18. D. Guillarme and M.W . Dong (Eds.), Trends in Anal. Chem. 63, 1–188 (2014).
19. V. D’Atri, S. Fekete, A. Clarke, J-L. Veuthey, and Davy Guillarme, Anal. Chem. 91(1), 210–239 (2019).
20. J. De Vos, K. Broeckhoven, and S. Eeltink, Anal. Chem. (1), 262–278 (2016).
21. M.W. Dong, LCGC North Am. 35(6), 374, (2017).
22. M.W. Dong and D. Guillarme, LCGC North Am. 35(8), 486 (2017).
23. M.W. Dong, LCGC North Am. 35(11), 818–823 (2017).
24. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, ICH Q8(R2), Pharmaceutical Development (Geneva, Switzerland, 2009).
25. S. Fekete, J. Fekete, I. Molnár, and K. Ganzier, J. Chromatogr. A 1216(45), 7816–23 (2009).
26. E.F. Hewitt, P. Lukulay, and S. Galushko, J. Chromatogr. A 1107(1–2), 79–87 (2006).
27. R.P. Verseput and J. A. Turpin, Chromatography Today, Aug 26, 2015.
28. M. Wong, B. Murphy, J. H. Pease, and M. W. Dong, LCGC North Am. 33(6), 402 (2015).
29. M.W. Dong, LCGC North Am. 34(8), 540–545 (2016).
30. M.W. Dong, E.X. Zhao, D.T. Yazzie, C. C. Gu, and J.D. Pellett, Amer. Pharm. Rev. 15(6), 10–17 (2012).
31. S. Fekete, D. Guillarme, and M.W. Dong, LCGC North Am. 32(6), 420–433 (2014).
32. M.W. Dong and B.E. Boyes, LCGC North Am. 36(10), 752–767 (2018).
33. Analytical Procedure and Method Validation for Drugs and Biologics: Guidance for Industry (US FDA, CDER and CBER, Pharma Quality/CMC, Silver Spring, Maryland, (2015).
34. M.K. Parr and A.H. Schmidt, J. Pharm. Biomed. Anal. 147, 506–518 (2018).

Michael W. Dong is a principal of MWD Consulting, which provides training and consulting services in HPLC and UHPLC, method improvements, pharmaceutical analysis, and drug quality. He was formerly a Senior Scientist at Genentech, Research Fellow at Purdue Pharma, and Senior Staff Scientist at Applied Biosystems/PerkinElmer. He holds a PhD in Analytical Chemistry from City University of New York. He has more than 100 publications and a best-selling book in chromatography. He is an editorial advisory board member of LCGC North America and the Chinese American Chromatography Association. Direct correspondence to: LCGCedit@ubm.com​

Kim Huynh-Ba is the managing director of Pharmalytik LLC. (www.pharmalytik.com), which provides consulting services in stability sciences, quality management systems, and analytical development. She is an Adjunct Professor at Temple University-School of Pharmacy and Illinois Institute of Technology (IIT). Kim is a member of the US Pharmacopeia’s Council of Expert, chairing the Chemical Medicines Monograph IV Expert Committee, USP Good Documentation Practices Expert Panel, USP Organic Impurities of Drug Substance, and Drug Products Expert Panel. She is on the editorial board of the AAPS Open, the Journal of GXP Compliance, and the Journal of Validation Technology. Kim authored ~30 publications and edited two books on stability testing.

Joshua T. Ayers is the Principal Consultant at ASQ Solutions, LLC, which provides analytical, stability, and quality consulting services to the pharmaceutical industry. He has worked on several early and late-stage development projects and marketed products. He has managed analytical laboratories and stability programs for large pharmaceutical companies. Joshua holds a Ph.D. in Medicinal Chemistry from the College of Pharmacy, University of Kentucky. He has authored more than 20 publications and currently sits on the AAPS Stability Community.