Article pubs.acs.org/OPRD
Applying QbD Principles To Develop a Generic UHPLC Method Which Facilitates Continual Improvement and Innovation Throughout the Product Lifecycle for a Commercial API Jacky Musters,* Leendert van den Bos, and Edwin Kellenbach MSD Oss, GTO API, Molenstraat 110, 5342 CC Oss, The Netherlands ABSTRACT: The application of quality-by-design (QbD) principles for new chemical entities (NCEs) is highlighted in several publications. QbD, however, is rarely applied to marketed APIs. Lifecycles of existing APIs are extending. With this extension the focus on drug safety and compliance requires up-to-date knowledge of the product and its production process as over the years expectations in this respect have shifted. We applied QbD principles in order to set up an improved control strategy for the final five steps in the production route of a steroidal contraceptive, which has been produced for over 20 years within our facilities. A generic UHPLC method was developed for the quantitative analysis of the available intermediate batches which have all resulted in compliant API at full commercial scale. On the basis of the batch results, (statistically supported) specifications are proposed to create a range of proven acceptance criteria. This approach allows the application of the historical knowledge of the drug substance and its manufacturing process gained over the years to future production by exploiting the process capability of eliminating impurities and dealing with variability in the quality of the intermediates. Furthermore, it improves the process of specification setting. Generic UHPLC methods enable rapid quantitative monitoring of quality. The use of a generic UHPLC method in combination with the setup of a side-product flow scheme improves process understanding and supports chemical entity design space development. Furthermore, the use of a generic UHPLC method for multiple intermediates also offers the possibility to perform side-product tracing on the basis of retention times. A generic UHPLC method was developed according to QbD principles to create a range of proven acceptance criteria for the assay and side-product determination for the final five steps in the production route of a contraceptive API.
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INTRODUCTION “Quality-by-Design” is the concept of building quality into the product,1,2 or, as quoted in ICH harmonized tripartite guideline Q8,3 “The goal of manufacturing process development for the drug substance is to establish a commercial manufacturing process capable of consistently producing drug substance of the intended quality”. QbD principles can also be implemented within the process of developing an adequate control strategy, especially when a lot of process knowledge has been gained over the years or, as highlighted in ICH harmonized tripartite guideline Q11,4 “The control strategy might be developed through several iterations as the level of process understanding increases during the product lifecycle”. The analytical method development is a major contribution to the control strategy. On the basis of articles that have been published regarding the implementation of QbD principles in analytical method development,5−8 we defined QbD in analytical method development as “a systematic approach, beginning with predefined objectives, to obtain an increased scientific understanding and improved confidence in the final method developed, followed by continuous verification and improvement throughout the method lifecycle”. Several examples have been published in the literature that highlight the application of QbD principles for new chemical entities (NCE).9−11 By exploiting techniques such as design of experiments, process analytical technology (PAT) using sophisticated probes (FT-IR, Raman, etc.) and state-of-the-art scaled lab reactors, a thorough understanding of the manufacturing process is gained. Few examples of applications © 2012 American Chemical Society
of QbD to marketed products have been published. In our opinion there are two reasons for this. The first consists of the regulatory obstacles related to changing process parameters and updating analytical controls. The second is related to the investments required to perform these investigations. The lifecycles of existing APIs, however, are extending due to decreased approvals of NCEs12 and the use of existing APIs in novel formulations. The increasing focus on drug safety and compliance requires improved knowledge of the various impurities and their variances over the years. By applying QbD principles, an increased knowledge and understanding of the product and its production process is gained, which also improves the process of specification setting. Finally, a greater understanding of the product and its production process can create a basis for a more flexible regulatory approach.3,13 For commercial products a lot of knowledge has been gained over the years related to impurities, (process) chemistry, safety, etc. We applied this information to realize the implementation of QbD in order to develop an improved control strategy for the final five steps in the production route of a steroidal contraceptive. The steroidal compound has been produced for over 20 years within our facilities, and it takes >10 chemical conversions to produce this steroid from commercially available starting material of plant origin. Currently, the intermediates are released by thin layer chromatography (TLC). The TLC test performs a qualitative Received: October 17, 2012 Published: December 18, 2012 87
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potential side-product ranges measured by the method to be developed. We based the ATP on criteria for the API itself, currently released by HPLC. The objectives of the ATP are simply ‘What do you want the method to measure?’ and ‘How well does it have to measure it?’. In this case the analytical method should be suitable for the identification and accurate quantitative determination of the assay of the intermediates in the range of 80−120%, relative to the API concentration. The combined accuracy and precision of the method must be such that assay measurements fall within 100% ± 1%. The method should be able to separate all intermediates and known side products. Furthermore, the method should be able to perform an accurate quantitative determination of the content of the potential side products in the range of 0.05−1%, relative to the concentration of the active compound. The combined accuracy and precision of the method must be such that side-product content measurements fall within 100% ± 15% for levels ≤0.1% and 100% ± 10% for levels >0.1%. In the second step expressed as ‘Risk Assessment’ hazards are identified, and risks associated with exposure to those hazards are evaluated and eliminated.15 Examples of hazards are the availability of facilities, equipment, utilities, and analyst experiences. In preparation for the method development, also API characteristics (such as structures, volatility, solubility, UV sensitivity, etc.) were collected. The Risk Assessment step is mentioned after the ATP in Figure 1, since it is an indispensable step before selecting an analytical technique. However, a more precise view would be that Risk Assessment is applied to all steps in Figure 1 and will return throughout the method lifecycle to identify factors that can contribute to method performance and improvement. On the basis of the ATP, the analytical method should be suitable for the identification and accurate quantitative determination of assays and side-product contents. Chromatography is the preferred technique for method development, since compounds should first be separated before they can be detected and quantified. On the basis of previous experiences we knew that the intermediates are not thermally stable. GC was therefore not preferred. Since the intermediates and most of the side products are uncharged apolar molecules of a similar size (∼330 Da) the preferred technique to start with was reversed phase liquid chromatography. The intermediates and most of the side products contain chromophore groups. On the basis of these structure properties, diode array was selected as the detection technique. It was preferred to develop a generic state-of-the-art analytical method which can be introduced as a robust, reliable, and quantitative method for release testing within the QC laboratories. The analysis runtime should be at least equivalent in comparison with the current TLC methods, and health, safety, and environmental considerations should be taken into account. Furthermore, it is preferable to develop a method which is MS-compatible and offers the possibility to perform structure proposal or structure confirmation analysis on side products obtained. The liquid chromatography technique which can comply with all these requirements is UHPLC. A systematic approach was followed to efficiently develop a generic method for the steroidal intermediates. This approach involves the use of a computer-assisted commercially available chromatographic method development tool (ChromSword), which is capable of performing fully automated method development. Since our method development setup (see Figure
comparison of the samples against the reference standard. TLC is a simple and quick, low-cost, analytical technique which enables the analysis of multiple batches simultaneously. The interpretation of TLC may be difficult and analyst dependent (subjective). TLC has poorer separation efficiency in comparison, for example, to HPLC,14 and due to the qualitative manner it is not possible to perform trending. The main drawback, however, is the reference standard. This standard does not represent all acceptable impurity ranges because identity and levels of side products may vary batch-to-batch and are not covered by the reference standard. This may result in the rejection of intermediate batches which may have resulted in compliant API. The use of a quantitative and selective state-of-the-art analytical technique together with (statistically supported) specification setting should significantly increase the range of proven acceptance criteria for the intermediates. In addition, critical quality attributes (CQAs) of intermediates can be modified as product knowledge and process understanding increases. This contribution presents an improved analytical control strategy based on QbD principles.
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RESULTS AND DISCUSSION In the introduction we explained why it is crucial to investigate the possibilities of applying QbD principles within the manufacturing process of marketed APIs. In this section the analytical method development for the five final processes in the route towards the contraceptive API will be discussed. The aim of QbD in analytical method development is to design a robust and well-understood method that consistently delivers an adequate performance. The overall QbD process flow for analytical method development is depicted in Figure 1.
Figure 1. QbD process flow for analytical method development.
First we set up an analytical target profile (ATP). The ATP is based on the intended quality of the API, represented by the quality target product profile (QTPP). Critical quality attributes (CQAs) and critical process parameters (CPPs) for a given product and process, respectively, are used to achieve this QTPP.3,4 For our research we focused on assay and potential side-product CQAs. Other CQAs, such as, for example, appearance, morphic form, or particle size distribution, are not within the scope of this study since these CQAs are not controlled by the currently used TLC methods and also cannot be controlled by the developed ultrahigh-performance liquid chromatography (UHPLC) method. On the basis of extensive product knowledge gained over the years, it is known that the fate of potential side products is to be purged by the process. The available released intermediate batches which were used for method development and specification setting have all resulted in high-purity API with acceptable assays. Thus far, intermediates have been released by nonquantitative TLC methods. The assay and potential side-product CQAs for the intermediates will be expressed on the basis of the assay and 88
dx.doi.org/10.1021/op300292a | Org. Process Res. Dev. 2013, 17, 87−96
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Figure 2. Schematic diagram of the method development setup (adapted from ref 18).
2) contains an HPLC and not a UHPLC method, first an HPLC method was developed which was subsequently converted to a UHPLC method. By using ChromSword in combination with two external selection valves (one for the selection of eluents and one for the selection of columns) along with the prior knowledge and experience of the analytical scientists, the (U)HPLC method development time can be significantly shortened, and the probability of achieving optimum separation conditions can be enhanced.16−18 See Figure 2 for a schematic diagram of the method development setup. The development of a generic method for the intermediates was started by making a selection of columns and mobile phases for screening on the basis of the structural properties of the steroidal compounds and the potential side products. The selected HPLC technique to be used was reversed phase. Since neither of the compounds contains basic groups with a pKa value > 1.9 or acidic groups with a pKa value < 12.9, the mobile phase does not necessarily need to be buffered. The most common mobile phase compositions, combinations of acetonitrile/water and methanol/water, were tested. Since it was required to convert the developed HPLC method to a UHPLC method in a later stage, the column selection was limited to columns for which also a 2-sub-μm particle variant is available. Since the column selection was not limited to a pH range, both columns with a hybrid backbone as well as columns with a silica backbone were selected.19 Typical alkyl-bonded reversed phase columns (e.g., C18) do not always offer the required selectivity. Therefore, also a phenyl phase was added to the column set for screening.20 Phenyl phases can provide alternative retention characteristics, due to a type of electron donor−electron acceptor interaction between the π electrons of the phenyl ring of the bonded phases and the π electrons of an aromatic or polyunsaturated compound. Stationary phases containing shielded groups (polar embedded groups),21 which use an internal amide or carbamate group as the polar functionality, are particularly suitable for the separation of basic
compounds. The polar groups embedded in the bonded silane shield the free surface silanols from interaction with strong bases. Although the API and the intermediates do not have any basic properties, a stationary phase with shielding groups is generally part of the column set for screening since these types of stationary phases still might show an alternative selectivity. After suitable sample preparation, the combinations of columns and mobile phases were screened on HPLC using the isocratic fine optimization mode within the automated method development software ChromSword,22 see Table 1. Table 1. Columns and mobile phases selected for the screening experiment HPLC columns Xbridge C18, length 100 mm, diameter 3 mm, particle size 3.5 μm Xbridge Shield RP18, length 150 mm, diameter 3 mm, particle size 3.5 μm Xbridge Phenyl, length 150 mm, diameter 3 mm, particle size 3.5 μm Atlantis T3, length 150 mm, diameter 3 mm, particle size 3.5 μm mobile phases water methanol acetonitrile
The sample mixture was injected onto each column, and per column the isocratic fine optimization mode was run with both organic modifiers. The isocratic fine optimization mode is a method development mode in which mobile phase compositions are automatically selected and run by the software. The mobile phase composition starts at a manually selected level of the percentage of the organic modifier at which all components will elute. In this case the required level of organic modifier was not determined during earlier experiments; therefore, a level of 95% was selected (if the level is known, this will certainly reduce the screening time of the isocratic fine optimization). After the first run ChromSword calculates the number of peaks, peak resolutions, and retention factors. On the basis of these 89
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Figure 3. Overview of the optimal isocratic method conditions.
Figure 4. Chromatogram of a mixture of API and the potential side products. Labels: 1 = side product A; 2 = side product B; 3 = side product C; 4 = side product D; 5 = API.
converted to UHPLC conditions, and further optimization was done on the UHPLC system using columns with 2-sub-μm particles. The small-particle technology23,24 provides not only increased efficiency but also the ability to work at increased linear velocity without a loss of efficiency, providing both resolution and speed.25,26 The narrower the peaks are, the easier they are to separate from each other. Also, peak height is inversely proportional to the peak width; thus, as the particle size decreases, an increase in sensitivity is obtained, since narrower peaks are taller peaks.27 The intermediates III and IV are semi-pure variants of the API, which differ only by the presence of side products. A gradient was introduced for the elution of the apolar components to speed up the analyses and to improve the sensitivity. The optimal flow was determined by increasing the flow to a level at which the system pressure does not exceed the critical point of 1000 bar for the most viscous mobile phase composition (in practice, the maximum pressure was set around 850 bar). The optimal injection volumes and sample concentrations were determined in relation to the desired limit of quantitation for the potential side products. The final method conditions after optimization are shown in Table 2. A chromatogram of a sample of a mixture of API, the intermediates I and II (all at an equal level of 0.25 mg/mL), and potential side products (at a 1% level) is shown in Figure 5. For fine-tuning the method conditions and to verify the robustness of the generic method, a full factorial design of experiments (DoE) was used in which the parameters, column
results, the mobile phase composition is automatically adopted by the ChromSword software. After each run, the results of the actual and prior runs are automatically compared, and the software decides to continue optimizing or concludes that the optimal mobile phase composition is obtained. The goal of the isocratic fine optimization mode is to develop a method with a minimal peak resolution of 2 for all components and a maximum retention factor of 10 for the latest-eluting component. The selection of the best column and mobile phase composition is made by the analyst. The selection is subjective, but straightforward. The best conditions are chosen on the basis of the number of detected or separated peaks, peak shape, and total run time. On the basis of the results of the ChromSword screening, the highest resolution within the shortest time was obtained using an Xbridge Shield RP18 column in combination with an acetonitrile/water mobile phase. A chromatogram obtained at these isocratic conditions (see Figure 3) is shown in Figure 4. The intermediates I and II have more apolar structural properties and will not elute within an acceptable analysis time using a water/acetonitrile 55:45 v/v % mobile phase. A gradient should be introduced for the determination of these apolar intermediates and potential side products. To sufficiently separate the API, the intermediates, and the potential side products within a generic HPLC method, run times have to be increased significantly, and the sensitivity for late-eluting components will be decreased as a result of peak broadening effects. Therefore, the HPLC method conditions were 90
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Table 2. Final method conditions after optimization on UHPLC column
Table 3. Varied parameters in full factorial DoE
Acquity BEH Shield RP18, 100 mm × 2.1 mm; i.d. 1.7 μm 40 °C 95:5 v/v% water/acetonitrile 95:5 v/v% acetonitrile/water
column temp. eluent A eluent B Mobile Phase time (min) eluent A (%) 0 75 1 75 11.5 5 12.5 5 12.6 75 17 75 flow (mL/min) 0.8 detection (UV, nm) 244 injection vol (μL) 1
eluent B (%) 25 25 95 95 25 25
factor 1
factor 2
factor 3
run
column temp. in °C
flow in mL/min
% acetonitrile in eluent B
1 2 3 4 5 6 7 8 9 10 11
40 50 50 30 30 40 30 30 50 50 40
0.8 0.9 0.7 0.9 0.7 0.8 0.9 0.7 0.7 0.9 0.8
95 92 92 92 92 95 98 98 98 98 95
acetonitrile in eluent B the resolution of the API decreases faster when decreasing the column temperature compared to the upper level of acetonitrile in eluent B, which is shown by two nonparallel lines in the interaction plot which will finally intersect. When varying the flow in combination with the percentage of acetonitrile in eluent B, there was no or hardly any interaction with regard to the resolution factors. When varying the column temperature in combination with the flow or the percentage of acetonitrile in eluent B, interactions with regard to the resolution factors were obtained for several peaks. However, under all tested conditions the resolution factors are acceptable. Figure 7 represents interaction plots for the API. On the basis of the results from the full factorial DoE, the method is considered to be robust; method parameters can be varied in a sufficient range. The method conditions are robust and close to the optimum of the DoE. The optimal method conditions based on the DoE results would be the conditions as described in Table 2 in combination with a column temperature of 30 °C. However, at a temperature of 30 °C, the increase of the column pressure is unacceptable. With the conditions as mentioned in Table 2, intermediate I, intermediate II, the API, and the potential side products are baseline separated. In addition, 30 °C represents the extreme of the robustness range, and it would be more adequate to select conditions inside this range to ensure that small variations fall inside the studied
temperature, mobile phase composition, and flow, were varied (Table 3).a The API and its four intermediates were analyzed at the different design conditions. Retention times and resolution factors of the API, the intermediates, and the potential side products were compared. No or hardly any interaction was obtained between the factors tested with regard to the retention time. To illustrate this, Figure 6 represents interaction plots for the API. A full factorial design contains all possible combinations of low/high levels for all the factors. In an interaction plot twofactor interactions were confounded with one another. For example, in the first plot of Figure 6, the effect of interactions of the factors (A) column temperature and (B) flow on the retention time of the API is shown. Both at the upper and lower flow levels tested, the retention time of the API decreases, when the column temperature is increased. There is no interaction between flow and column temperature regarding the retention time of the API, which is shown by two parallel lines in the interaction plot which do not intersect. In the second plot of Figure 7 the effect of the interactions of the factors of (A) column temperature and (C) percent of acetonitrile in eluent B on the resolution of the API is shown. At the lower level of
Figure 5. Chromatogram of a mixture of the API, the intermediates I and II, and the potential side products at UV 244 nm. Labels: 1 = side product E*; 2 = side product A; 3 = side product B; 4 = side product C; 5 = side product D; 6 = API; 7 = intermediate I; 8 = intermediate II. *Side product E is a side product which is present in intermediate I. 91
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Figure 6. Interaction plots for the retention time of the API.
As shown in Figure 8 the strength of the method lies in its generic character, which offers the possibility to perform side product tracing based on retention times. The chromatograms of subsequent intermediates along the route show at a glance the formation, fate, and purge of potential side products through every step of the manufacturing process. However, side products can also convert. Therefore, it is advisable to use additional techniques such as, for example, mass spectrometry and multidimensional, heteronuclear NMR to perform full sideproduct tracing. As mentioned in the introduction, drawbacks of the currently used TLC methods are the subjective interpretation and the qualitative determination of the purity against a reference standard which does not represent all acceptable impurity ranges. This may result in the rejection of intermediate batches which would have resulted in compliant API. In contrast, using a set of representative intermediate batches (which have all resulted in compliant API) to set specifications on side products by using an objective, quantitative technique will create a range of proven acceptance criteria encompassing all the qualities of batches used in specification setting. This approach thus allows applying the process and (historic) product knowledge gained over the last 20 years for future production by exploiting the process capability to eliminate impurities and deal with variability in the quality of the intermediates. For the specification setting of each individual intermediate, a set of representative batches (which have all resulted in compliant API at full commercial scale) was analyzed. An
robustness intervals. Therefore, the column temperature of 40 °C was unaltered. Outside the design,column robustness, linearity, and sensitivity were also verified. The method is robust using different column batches of the same type (same vendor, serial number, and dimensions). The API, the intermediates, and the potential side products are baseline separated on each of the tested columns, and the variations in retention times are minor (
