Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 14) docx

14 ROLE OF HPLC IN PROCESS DEVELOPMENT Richard Thompson and Rosario LoBrutto 14.1 RESPONSIBILITIES OF THE ANALYTICAL CHEMIST DURING PROCESS DEVELOPMENT In the drug discovery area, a compound with desired therapeutic properties is identified, and its structure may be modified by synthetic alterations to enhance potency and specificity or to decrease toxicity and undesired side effects The lead drug candidate is then transitioned into the drug development area Only small amounts of drug (typically less than a gram) are required to support the required studies in the Drug Discovery area However larger amounts are required to support the studies conducted in the Drug Development area The amount required in the preclinical stage typically ranges from 20 to 2000 g This material is required to support studies including subchronic toxicity, genotoxicity, ancillary pharmacology, early animal pharmacokinetics (PK), salt/form selection, and formulation development As the drug candidate progresses through the various clinical stages, the drug requirements typically range from kg to 200 kg This material supports the various clinical studies as well as chronic toxicity, carcinogenicity, development and reproductive toxicity, and formulation development Finally, tons of drug may be required upon successful approval and commercialization (Figure 14-1) The synthetic pathway to the drug substance is likely to evolve during the various stages of development It is highly unlikely that the synthetic process HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc 641 642 ROLE OF HPLC IN PROCESS DEVELOPMENT Figure 14-1 Stages of process development in the context of drug development utilized in drug discovery will be the same as that used to provide commercial scale quantities The discovery chemist may utilize a large number of synthetic steps, use a number of reagents that are expensive or not practical at scale-up, use a number of chromatographic steps for purification, and experience very low yields For scale up, the process development chemist must factor in safety, economical, and ecological considerations while producing a robust and reproducible synthesis He must consider operating limitations such as heat and mass transfer Economic factors will dictate minimization of the synthetic steps, maximization of yield, and choice of raw materials In addition, the process must meet environmental, occupational health, and safety requirements Furthermore, the process development chemist must follow guidelines from the Food and Drug Administration (FDA) in relation to the control and identification of impurities in drugs that will be used in humans Regulatory bodies require that the maximum possible human exposure to an impurity in a drug substance be supported by toxicological studies in animals that indicate no significant adverse effects Consequently, impurities that exceed a 0.1% tolerance limit in clinical material must first be qualified in animal toxicological studies Scale up of a synthesis, however, may generate a different impurity profile than observed for the smaller quantities prepared to support the toxicological studies Kinetic factors, changes in raw materials, or changes in reaction conditions may result in the introduction of new or elevated impurities These new impurities may be qualified in additional chronic toxicity and genotoxicity studies, but this strategy is often not economically feasible and is undertaken more as a last resort A better strategy is to identify and then control impurities that are generated during the continuously evolving stages of process development HPLC SEPARATION MODES 643 As a consequence of process evolution and regulatory requirements, the analytical chemist supporting process development is faced with a number of challenges He must evaluate the purity and stability of raw materials, intermediates, and drug substance He must evaluate yield and impurity generation across the various synthetic steps Impurities in drug substance, intermediates, and raw materials may require identification Analytical methods may have to be adapted to accommodate process changes Finally he must set specifications, validate analytical methods, provide regulatory documentation, and perform a technology transfer prior to drug approval and commercialization To this end, HPLC is a critical tool to perform many of the above tasks Most pharmaceutical compounds are amenable to analysis by HPLC HPLC is a powerful technology that is capable of separating complex mixtures into individual components that can then be quantified A well-developed HPLC method resolves and quantifies impurities from an analyte of interest in a reproducible, rugged, precise, and accurate fashion 14.2 HPLC SEPARATION MODES The multitude of available separation modes, mobile phases, and columns provide a plethora of parameters that can be manipulated to meet the criteria for a well developed HPLC method Conversely, it also creates a dilemma in choosing the optimal parameters from the myriad of possibilities The commonly utilized modes of HPLC in pharmaceutical development are reversed phase (RP) and normal phase (NP) for small organic molecules (10% degradation is observed in less than an hour, the solutions may be put in the refrigerator to slow down the kinetics of the degradation A similar procedure is employed for oxidative stress using 3% hydrogen peroxide as the starting point Thermal stability is demonstrated by holding solid sample at ∼10°C below the melting point of the compound, in a GC oven, until significant degradation is observed by the HPLC method If thermogravimetric analysis or differential scanning calorimetry indicates that significant degradation occurs below the melting point, a lower temperature may be chosen to conduct the study Photodegradation is generally performed with the drug substance in solution kept in a clear glass container on a benchtop exposed to the normal light source in the lab or placed in a qualified light chamber (i.e., cool white fluorescent and near-ultraviolet lamp) 14.8.1 Stability in Solution and Forced Degradation Studies (Process Intermediate Compound A) In the following case study, during the course of the initial method development for an intermediate, some late eluting species (18–20 minutes) were presumed to be forming in the sample solutions (prepared in acetonitrile) in ESTABLISHMENT OF HPLC SELECTIVITY BY STRESS STUDIES 667 Figure 14-11 w pH study on Luna C18 (2) for intermediate compound A Method w conditions are indicated in the figure ambient conditions under normal room light In Figure 14-11 (w pH 7, column w temperature 30°C, for sample stored for 36 hours at room temperature) these potential degradation products formed in solution are circled Further studies were performed in order to determine whether these degradation products originate from interactions with the diluent and/or are unstable in the diluent under normal light conditions at ambient conditions in the laboratory Two samples of the intermediate were prepared using acetonitrile and methanol as the diluents Each sample preparation was immediately analyzed using an autosampler-tray-cooler-equipped HPLC for the initial control timepoint, and portions were also stored under ambient conditions (clear volumetric flask and ambient room lighting) and under refrigerated conditions for 50 hours The samples prepared in acetonitrile and stored at ambient temperature showed increased levels of three major impurities (RRT = 1.76–1.78, RRT = 2.06, RRT = 2.10–2.11 Figure 14-12) The two impurities at RRT 2.06 and RRT 2.10–2.11 were not present in the refrigerated solution.API-ESI LC-MS analysis of these degradation products suggested that the RRT = 1.76–1.78 was a desN-oxide impurity [M − 16 + H].The RRT = 2.06, and RRT = 2.10–2.11 impurities had the same mass [M + 18 + H] and were presumed to be N-hydroxy adducts (OH group added across one of the double bonds on the ring) of the intermediate.These findings were further confirmed by using deuterated mobile phases which indicated the presence of an additional labile hydrogen atom With methanol as the diluent, the peaks eluting at RRT = 2.06 and RRT = 2.10–2.11 were not observed However, other degradation products were formed (Figure 14-13) The formation of these additional degradation 668 ROLE OF HPLC IN PROCESS DEVELOPMENT Figure 14-12 Stability study in acetonitrile, ambient (normal room light) versus refrigerated conditions Figure 14-13 Stability study in methanol, ambient versus refrigerated conditions products in the methanol diluent was also inhibited at 4°C and was at lower levels than the degradation products formed in acetonitrile diluent Two major impurities found when using the methanol diluent, RRT = 2.38 and RRT = 2.18, were identified API-ESI LC-MS analysis of these degradation products suggested the RRT = 2.38 and the RRT = 2.18 are [M + 32 + H] species and are presumed to be N-methoxy adducts of the intermediate These degradation products are at much lower levels than the degradation products formed in acetonitrile These studies showed that this intermediate is more ESTABLISHMENT OF HPLC SELECTIVITY BY STRESS STUDIES 669 Figure 14-14 Overlay of light-stressed sample in acetonitrile stable in methanol than in acetonitrile; however, in both solvents, the sample solutions are stable in the refrigerator (4°C) for up to 50 hours Solution light stability studies (dark and light chamber) in both solvents at ambient temperature were also performed in order to further determine whether the formation of the degradation products is photocatalyzed or generated at ambient temperature (∼25°C) Control samples were placed in an amber flask at ambient conditions, and the stress samples were placed in a light chamber employing white-light intensity and ultraviolet-light intensity at room temperature The solutions were then analyzed after approximately 0, 21, 49, and 140 hours (Figures 14-14 to 14-16) The samples exposed to light in both diluents showed considerable degradation over the course of the study, while the samples protected from light at room temperature remained stable for up to 140 hours As a result of this study, the method was modified to prescribe that all samples be prepared in amber glassware using methanol diluent Having established that the sample preparations are light-sensitive, it was necessary to also establish if the solid was light sensitive A light stress study with a “pure” sample (recrystallized intermediate) was carried out over 141 hours where a sample of API solid was placed in a quartz glass dish and left in the light chamber at ambient conditions A sample was analyzed at the following time points: 0, 24.5, 67.5, 113, and 141 hours A control sample was placed in a similar dish and protected from light with aluminum foil This sample was placed in a dark cabinet and analyzed after 143 hours The solid under the light stress conditions showed significant degradation over the course of the study (total impurities grew from 1.3% to 2.2%), while the control sample stored in the dark for at least 143 hours demonstrated no significant growth in impurities The intermediate should have a protect-from- 670 ROLE OF HPLC IN PROCESS DEVELOPMENT Figure 14-15 Overlay of light-stressed sample in methanol Figure 14-16 Overlay of dark control samples (stored in amber flasks) in methanol and acetonitrile: T = versus T = 140 hr light requirement, and sample preparation should be performed in amber (Actinic) glassware with methanol diluent in order to prevent potential lightinduced degradation products 14.9 HPLC METHOD VALIDATION Following establishment of selectivity, the HPLC method should be validated The validation of an analytical method is meant to demonstrate the suitability of the method to perform its intended purpose Validation is an important HPLC METHOD VALIDATION 671 portion of the filed regulatory documentation to support an IND or an NDA [46–48] ICH guideline Q2A defines the validation terms and Q2B describes methods for the determination of various validation parameters including specificity, linearity, accuracy, precision, detection and quantitation limits, robustness, and system suitability testing [47, 48] The level of validation is stage-dependent, and the validation criteria increase as the projects evolves from preclinical to clinical to commercialization While these requirements pertain particularly to the regulatory HPLC impurity profile, any HPLC method should be validated to some level to ensure acceptable accuracy and precision 14.9.1 Prevalidation and System Suitability Prior to performing a formal validation, the analytical chemist should have performed some prevalidation during method development The expectation is that a well-developed HPLC method should subsequently be validated with no major surprises or failures Prior to validation, specificity and some degree of robustness should be demonstrated In addition, some form of system suitability criteria will have been established System suitability evaluates the capability of an HPLC system to perform a specific procedure on a given day It is a quality check to ensure that the system functions as expected and that the generated data will be reliable Only if the system passes this test should the analyst proceed to perform the specific analysis System suitability can be based on resolution of two specified components, relative standard deviation, tailing factor, limit of quantitation or detection, expected retention times, number of theoretical plates, or a reference check System resolution depends upon resolution achieved between the peak for the drug substance and the peaks directly preceding and succeeding it The lowest resolution between any pair of peaks is also critical A minimum resolution can be set for two peaks to establish system suitability For assays, a maximum relative standard deviation for multiple injections of a standard can be used as a system suitability criterion Minimum theoretical plates, a maximum tailing factor, a retention time range for a specific peak, and the ability to visually observe a peak for a 1000-fold dilution of the normal injection concentration are all appropriate system suitability criteria Alternatively, a reference standard sample containing several low-level impurities can be injected, and various parameters such a resolution, retention time, and the ability to detect certain low level impurities can be used to establish system suitability Also, if a sample is not available that contains an impurity that is known to form during stability, this impurity can be generated under stress conditions and used as a system suitability solution For example, if a drug substance is known to oxidize readily at a particular moiety and the potential impurity elutes close to the active ingredient, the impurity can be generated with a suitable stress solution for a defined period of time and a critical resolution criterion can be set 672 ROLE OF HPLC IN PROCESS DEVELOPMENT 14.9.2 Validation Once the method is determined to be optimum and system suitability criteria has been set, validation can be initiated There may be different stages of validation, and this can vary from one pharmaceutical company to another For example, some companies may have a three-tier validation strategy (validation testing to be available prior to the first drug substance delivery, validation to be available for regulatory methods when IND is filed, validation to be available for regulatory methods to be transferred to the manufacturing facility and to support the NDA filing) or a two-tier validation strategy (early phase before final synthesis is set and late phase after final synthesis is set) Specificity is demonstrated through the resolution of the drug substance from impurities Selectivity from starting materials, intermediates, by-products, and degradation products is a good starting point to demonstrate specificity Stress studies and peak purity experiments using diode array analysis or MS can suffice for selectivity at the early stage of development At later stages of development, isolated impurities will become available They can then be utilized to establish selectivity and low-level linearity, and to determine their relative response factors Peak purity can be assessed by visual examination of the peaks for Gaussian symmetry, by diode array or MS, or by use of an orthogonal LC method The stability of the sample solution should also be evaluated as a function of time and storage temperature A suitable criterion is that sample solutions should be stable for at least 24 hours under defined storage conditions (e.g., amber glassware, low temperature) If greater solution stability is desired, the solution stability at refrigerated conditions should be compared to the solutions stored at ambient conditions Linearity (typically correlation greater than or equal to 0.999) can be established from the limit of quantitation (LOQ) to 120% or 130% of injection concentration (minimum five serial dilutions) or segmented into low (e.g., LOQ to 1%, minimum three concentrations) and high concentration (e.g., 50–130%) linearity Establishment of LOQ and limit of detection (LOD) is also critical for early stage validation There is flexibility in setting LOQ and LOD LOQ and LOD is generally established at a minimum signal-to-noise ratio (SNR) of 10 and 3, respectively The target LOQ is generally 0.05% (one-half of the identification threshold defined in the ICH 3A guidelines) Other criteria that can be set are (a) a maximum deviation of 10–20% of the response factor of the LOQ solution compared to a 5X LOQ solution and (b) a maximum 10–15% RSD for area counts for a minimum of three injections for the LOQ solution The accuracy of the method can be determined by performing recovery experiments or by comparison to another analytical method (such as titration, DSC, and PSA) Spiking experiments, where increasing amounts of an impurity are introduced into the sample and the accuracy of the result versus TECHNOLOGY TRANSFER 673 theoretical is evaluated, are usually conducted at later stages of development where authentic isolated impurities become available When the assay is being used for a minor component, recovery should be performed for at least three concentrations ranging from 120% of the impurity specification to the LOQ The relative response factors of the impurities compared to the active drug substance should also be determined at this stage as well Validation of the precision of an HPLC method occurs at three stages The first stage is injection precision based on multiple injections of a single preparation of a sample on a particular sample on a given day The second stage is repeatability where multiple preparations of a sample are analyzed with multiple injections by the same chemist on the same day The third stage requires analysis of multiple preparations by more than one analyst, on different instruments on different days Robustness is also demonstrated at later stages of development and reflects the ability of the method to remain unaffected by minor variations in operating conditions such as injection amount, flow rate, column temperature, mobile-phase composition, and for different lots of columns 14.10 TECHNOLOGY TRANSFER The final step in process development for the analytical chemists is the transfer of his methods to the manufacturing division This transfer usually occurs late in clinical phase III and prior to filing the NDA The transfer is conducted to ensure that the method can be implemented and used correctly in the new lab The method transfer serves as training for the receiving lab and requires that the receiving lab demonstrate the capability to perform the method At this stage, method validation would be completed, although some companies may involve the receiving lab in the validation process as part of the transfer Validation performed by the receiving lab qualifies it to use that method for its intended purpose The method transfer is executed through the authoring of a method documentation package that should include a detailed description of the method, some insight into the method development process, a validation summary, and a transfer protocol The transfer protocol should clearly define the role and responsibilities of the transferring and receiving groups It should list all of the instrumentation, columns or equivalent columns, chemicals, and samples required to run the method, outline the experiments that need to be performed, and the acceptance criteria for successful transfer of the method The transfer documentation must be reviewed and agreed upon by both groups Upon completion of the method transfer, data are compiled and analyzed and a final report is written This report should indicate whether the method was successfully transferred and should list any deviations that were made to the original protocol 674 ROLE OF HPLC IN PROCESS DEVELOPMENT 14.11 CONCLUDING REMARKS HPLC plays a significant role in the analytical aspect of process development It is the most commonly used tool to determine the purity of the active pharmaceutical ingredient and to track impurity generation and yield during the process There are a plethora of options available in terms of separation mode, stationary phase, and mobile phase to cover most of the wide range of diverse physiochemical properties associated with the active pharmaceutical ingredients, raw materials, intermediates, and impurities This chapter has presented some of these options and how they can be applied Procedures to develop HPLC methods and to ensure that the methods are precise and accurate have also been presented Finally, how these HPLC methods fulfill regulatory requirements and how they are successfully transferred to manufacturing sites have been outlined REFERENCES J Xu, R Thompson, B Li, and Z Ge, Application of packed column supercritical fluid chromatography for separation of bromosulfone from process related impurities, J Liq Chrom Relat Technol 25 (2002), 1007–1018 M Yang and R Thompson, A retention and selectivity model for hydrophilic interaction chromatography HILIC, submitted to J Chrom A (Aug 2006) Q Tu, T B Wang, X Jia, and X Bu, Speciation analysis of halogenides and oxyhalogens by ion chromatography with inductively coupled plasma mass spectrometer as element-specific detector, Merck Research Laboratories internal communication R Thompson, N Grinberg, H Perpall, G Bicker, and P Tway, Separation of organophosphonates by ion chromatography with indirect photometric detection, J Liq Chrom 17 (1994), 2511–2531 M See, R Thompson, N Grinberg, H Perpall, G Bicker, and P Tway, Chromatographic analysis of residual acetate in bulk drugs, J Liq Chromatogr 18 (1995), 137–154 FDA’s policy statement for the development of new stereoisomeric drugs, Chirality (1992), 338 P Borman, B Houghtflower, K Cattanach, K Crane, K Freebairn, G Jonas, I Mutton, A Patel, M Sanders, and D Thompson, Comparative performances of selected chiral HPLC, SFC, and CE systems with a chemically diverse sample set, Chirality 15 (2003), S1–S12 C Welch, Evolution of chiral stationary phase design in the Pirkle laboratories, J Chromatogr A 666 (1994), 3–26 R Thompson, Z Ge, N Grinberg, D Ellison, and P Tway, Mechanistic aspects of the stereospecific interaction for aminoindanol with a crown ether column, Anal Chem 67 (1995), 1580–1587 10 R Thompson, unpublished data REFERENCES 675 11 T Ward and A Farris III, Chiral separations using the macrocyclic antibiotics: A review, J Chromatogr A 906 (2001), 73–89 12 J Haginaka, Protein-based chiral stationary phases for high-performance liquid chromatography enantioseparations, J Chromatogr A 906 (2001), 253–273 13 R.Thompson,V Prasad, N Grinberg, D Ellison, and J.Wyvratt, Mechanistic aspects of the stereospecific interactions of immobilized a1-acid glycoprotein, J Liq Chrom Relat Technol 24 (2001), 813–826 14 E Yashima, Polysaccaride-based chiral stationary phases for high-performance liquid chromatographic enantioseparation, J Chromatogr A 906 (2001), 105–125 15 K Tachibana and A Ohnishi, Reversed-phase liquid chromatographic separation of enantiomers on polysaccharide type chiral stationary phase, J Chromatogr A 906 (2001), 127–154 16 T O’Brien, L Crocker, R Thompson, K Thompson, P Toma, D Conlon, B Feibush, C Moeder, G Bicker, and N Grinberg, Mechanistic aspects of chiral discrimination on modified cellulose, Anal Chem 69 (1997), 1999–2007 17 H Ding, N Grinberg, R Thompson, and D Ellison, Enantiorecognition mechanisms for derivatized cellulose under reversed phase conditions, J Liq Chrom Relat Technol 23 (2000), 2641–2651 18 Y Bereznitski, R LoBrutto, and N Grinberg, Trace analysis of sodium 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nitrogen detector for analyzing compounds lacking a sufficient UV chromophore, LC-GC North Am 21 (2003), 376–381 25 R Thompson, unpublished data 26 D Robb, T Covey, and A Bruins, Atmospheric pressure photoionization: An ionization method for liquid chromatography-mass spectrometry, Anal Chem 72 (2000), 3653–3659 27 M Reta, P Carr, P Sadek, and S Rutan, Comparative study of hydrocarbon, fluorocarbon, and aromatic bonded RP-HPLC stationary phases by linear solvation energy relationships, Anal Chem 71 (1999), 3484–3496 28 R Kaliszan, M van Straten, M Markuszewski, C Cramers, and H Claessens, Molecular mechanism of retention in reversed-phase high-performance liquid 676 29 30 31 32 33 34 35 36 37 38 39 40 41 42 ROLE OF HPLC IN PROCESS DEVELOPMENT chromatography and classification of modern stationary phases by using quantitative structure–retention relationships, J Chromatogr A 855 (1999), 455–486 R Vervoort, A Debets, H Claessens, C Cramers, and G de Jong, Optimisation and 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DeTora, T Maher, P McKenzie, and D Ellison, On-line HPLC monitoring of a deprotecting process, J Process Anal Chem (1997), 1–6 H Minakuchi, K Nakanishi, N Soga, N Ishizuka, and N Tanaka, Effect of skeleton size on the performance of octadecylsilylated continuous porous silica columns in reversed-phase liquid chromatography, J Chromatogr A 762 (1997), 135–146 N Wu, J Dempsey, P Yehl, A Dovletoglou, D Ellison, and J Wyvratt, Practical aspects of fast HPLC separations for pharmaceutical process development using monolithic columns, Anal Chim Acta 523 (2004), 49–156 ICH, Q3A: Impurities in new drug substances, 1996 ICH Guideline: Impurities in new drug substances, Federal Register 61 (1996), 371ff International Conference on Harmonisation Steering Committee Revised Guidance on “Impurities in new drug substances,” Federal Register 68 (2003), 6924–6925 REFERENCES 677 43 K Albert (ed.), On-Line LC-NMR and Related Techniques, John Wiley & Sons, Chichester, Sussex, UK, 2002 44 N Nyberg, H Baumann, and L Kenne, Application of solid-phase extraction coupled to an NMR flow-probe in the analysis of HPLC fractions, Magn Reson Chem 39 (2001), 236–240 45 International Conference on Harmonisation Steering Committee, Stability testing of new drug substances and products, 1999 46 CDER Guideline on Validation of Chromatographic Methods, Reviewer guidance of chromatographic methods, US Food and Drug Administration, Center for Drugs and Biologics, Department of Health and Human Services, 1994 47 ICH, Q2A: Validation of analytical methods: Definitions and terminology, October 1994 48 ICH, Q2B: Analytical validation—methodology, November 1996 ... documentation, and perform a technology transfer prior to drug approval and commercialization To this end, HPLC is a critical tool to perform many of the above tasks Most pharmaceutical compounds... ROLE OF HPLC IN PROCESS DEVELOPMENT Electrochemical detection can be utilized for compounds that are ionic or readily oxidizable or reducible Thus, this form of detection can be used for the... column w temperature 30°C, for sample stored for 36 hours at room temperature) these potential degradation products formed in solution are circled Further studies were performed in order to determine