A Guide to Effective Method Development in Bioanalysis

A Guide To Effective Method Development In Bioanalysis-Free PDF

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A G uide to E ffective M ethod D evelopment I N B I OA N A LYS I S. Quantitative Drug Analysis and Pharmacokinetics Make a Synergistic Partnership 3. Bioanalysis From Vial to File 9, DEVELOP MS MS METHOD Robust MRM MS MS Method Development for Bioanalytical Assays 17. Evaluate MS response and develop,a multiple reaction monitoring. methodology that provides the,optimum MS conditions for detection. of the compounds of interest, DEVELOP LC METHOD Strategic LC Method Development for Bioanalytical Assays 23. Robust chromatography will ensure,the analyte s of interest separate.
from both endogenous interferences,and drug metabolites. Dev elop Sam ple Sample Preparation in Method Development for Bioanalytical Assays 31. P reparation Met hod,Preparing the sample for analysis. is critical to ensuring that the assay,conforms to recognized standards of. compliance and performance Analytical,performance whether sensitivity. reproducibility or throughput,depends on a clean sample.
Informatics in Bioanalytical Laboratories 39, The Waters ACQUITY UPLC System Increasing Productivity. and Profitability in Quantitative Bioanalysis 43, The Waters ACQUITY UPLC MS MS System Increasing Efficiency. Productivity and Profitability for Bioequivalence Laboratories 47. Key Waters Technologies 49,Quantitative drug analysis and pharmacokinetics. mak e a syne rgistic pa rtne rship,John Ayrton Ph D GlaxoSmithKline retired. Developments in bioanalytical technology and the,DR JOHN AYRTON.
application of pharmacokinetic PK principles have created. Dr John Ayrton is a consultant who has 35 years, a synergistic partnership that plays a vital influential of experience in pharmaceutical R D. role in the discovery and development of new medicines After graduating in chemistry Ayrton joined. GlaxoSmithKline in London as a research scientist in. The origins of this partnership can be traced back some drug metabolism His initial work in that department. 30 years and an understanding of the way in which the had a major impact as he set up the first HPLC system. to be used for the assay of drugs in biological, application of these two scientific disciplines has evolved samples This work in the mid 1970s started a. lasting interest in bioanalysis this interest is evident. is helpful in ensuring we make optimal use of current in Ayrton s publications list which includes more. analytical technology than 20 applications of LC MS or LC NMR in drug. disposition studies,During the early 1980s Ayrton was awarded a Ph D. Whilst the principles of pharmacokinetics PK the study of the change. for his research on beta lactam pro drugs He also, of drug concentrations with time were first defined in the 1960s the became a Fellow of The Royal Society of Chemistry. application of PK in optimizing drug therapy and evaluating bioavail An appointment as head of Drug Metabolism. ability was truly made possible by the skills of the analytical chemists and Pharmacokinetics at GlaxoSmithKline in. who pioneered the development of HPLC in the 1970s 1987 led to other senior management roles in. pre clinical development and bioanalysis and to, During the 1980s HPLC UV based assays routinely provided the project leadership of Drug Discovery and Product.
plasma concentration data that were used to define drug exposure in Development teams. test animals and in human subjects the two main parameters of drug Ayrton s project team work brought direct. exposure being area under the plasma concentration time curve AUC involvement in 10 successful new medicines for. and maximum plasma concentration C max the treatment of bacterial viral or respiratory. diseases He also gained a wealth of experience in, At this time PK was applied mainly as a descriptive science drug development from the compounds that failed. essentially defining what happened to the test drug when it was during early clinical studies. administered to an animal in toxicology or to a healthy human. In December 2006 Ayrton retired from his Vice, subject in Phase I clinical pharmacology Drug safety and tolerability President role in project leadership at GSK but. plus the determination of PK parameters like plasma clearance he continues to work in drug discovery now with. volumes of distribution elimination half life and bioavailability smaller companies in the United Kingdom and. were and still are the objectives of Phase I studies United States This has helped him to maintain. a keen interest in the events and science of the,more pharmaceutical industry. The relationship between AUC and Cmax and key PK,Calculation of key. parameters such as plasma clearance bioavailability and PK parameters. bioequivalence are shown in Figure 1 in order to illustrate C 0 Concentration at T 0. how PK principles are applied at various stages of the Cmax V Volume of distribution. drug development process T 1 2 Half life, Over time it became established that poor PK profiles in.
new drug candidates accounted for clinical failure and. termination of the development of many compounds, Prentis et al Br J Clin Pharmacology 1988 25 387 96. In Phase I a poor PK profile would usually be character. ized by low and or highly variable oral bioavailability or T. a short elimination half life During later stages of clinical Figure 1 PK profile and Cmax. development when the drug is evaluated in the intended. patient population Phases II and III of drug development. the incidence of adverse side effects attributable to drug. The analytical technology of the early 1990s appeared to. metabolism or drug drug interactions also led to termina. have reached its limits but the commercial development of. tion of potential new medicines, atmospheric pressure ionization as an interface for LC. In all of these situations an understanding of the plasma heralded a revolution in bioanalysis Within a few years. clearance mechanisms of the test drug would have LC MS moved from being the method of last resort for. provided an early warning of potential problems with the quantitative analysis to being the method of choice the. compounds Furthermore such an understanding could hope that pharmacokinetic principles could be applied. potentially have helped to avoid the progression of a poor extensively as a tool in the lead optimization and candidate. drug candidate into clinical development studies and selection stages of drug discovery could at last be realized. helped to select an alternative compound with a superior. With the acceptance of LC MS MS as the gold standard. PK profile, technology to support PK and drug metabolism studies. Thus demand arose to expand the application of PK and comes significant improvements in assay sensitivity and. quantitative bioanalysis into lead candidate optimization specificity plus the potential to reduce assay time per. during drug discovery sample and consequently have a massive increase in the. demand for quantitative drug analysis, Progression to increased potency in pharmacology in the. late 1980s was already placing extra demands on HPLC UV Generating vast quantities of analytical data is much easier. based techniques Whilst the use of fluorescence detection and faster than ever consequently a basic grounding in. for LC and on rare occasions GC MS could be used pharmacokinetic principles is helpful in appreciating how. to improve assay sensitivity neither approach was widely bioanalytical data are to be used in drug discovery and. applicable for routine drug analysis The desire to apply development Here we will provide some examples of. PK principles to drug discovery would not only require an possible analytical strategies and an overview of the context. improvement in assay sensitivity significant increases in for applying these in selected phases of drug discovery. analytical capacity and faster sample turnaround would and development. also be needed,UPLC MS MS,elution SPE,pg mL 1 min,IV ITY UPLC MS MS.
25 pg mL 1 min,SE High Sensitivity,pg mL 5 min,GC MS LC MS MS. ng mL 30 min On line SPE,100 pg mL 10 min,HPLC UV ng mL 5 min. HPLC HPLC Fluorescence,WISP 500 ng mL SPE,HPLC UV 10 ng mL SPE. 10s g mL 40 min 25 min ng mL 25 min,1970 1980 1990 2000. The technology available for bioanalysis has evolved over the Most recently the application of a revolutionary new tech. last 30 years with an explosion in progress over the last 10 nique Waters UPLC Technology has allowed the industry. years due to the practical application of MS the evolution to further capitalize on the utility of MS and drive the limits. of column packings and novel solid phase extraction of sensitivity and speed of analysis to new levels. SPE techniques, Figure 2 The changing landscape of chromatography technology in bioanalysis.
Bioanalysis in t he drug discov e ry and dev elo pment lif ecyc l e. T he bioanalytical process, The lead optimization selection confirmation and testing process for new drug candidates is well defined as a series of. activities Broadly these can be split into discovery lead optimization and preclinical development through to clinical. evaluation Phases I to IV, Each stage places different requirements on the bioanalytical assay used to provide information The use of LC MS MS. assays provides the specificity flexibility and sensitivity to enable fast and effective decision making at each stage. PK and PK and bioanalysis in efficacy and safety studies. bioanalysis, Full PK characterization in Phase I First time into humans Phase IIa Proof of concept Phase III Long term studies. the drug discovery phase The key requirements for this The drug compound is tested Large numbers of patients take. is not required however stage are that the assay must fully in small groups of patients to part in Phase III clinical trials. in conjunction with in vitro characterize the absorption and assess efficacy in treating the with the objective of showing. techniques the ability elimination phases of the plasma disease state PK analysis is efficacy across a wide range of. to quickly assess the concentration time curve All employed to assess the dose populations Vast numbers of. bioavailability of a compound metabolites must be fully resolved exposure response PK PD This samples must be handled and. through bioanalysis gives identified and quantified is another key stage in deciding analyzed with a bioanalytical. a good indication of whether the drug should progress assay that is specific robust. suitability for advancement Adverse effects of a drug. further through clinical trials and and fast,to development toxicokinetics TK are investigated. therefore incur the investment n Assay specific to very. and require accurate measurement,required few analytes.
Some analytical priorities are of AUC and Cmax after single and. n Robust to variations in matrix, n Fast pass fail multiple doses In this way the Phase IIb. n Ability to process very large, determination of no toxic effect dose level can be Dose ranging studies are carried. volumes of data, PK parameters established a key parameter when out on patients to establish. n Medium sensitivity assay dosing in first time into human and effective doses for Phase II trials. n Minimum assay further trials,Analytical priorities include. development, The demands placed on the n High sensitivity assays.
n High specificity for the, bioanalytical assay are for n High specificity assays. compounds of interest, n High sensitivity to ensure that for drug compound and. the lowest effective doses can metabolites,be identified n Fast turnaround of samples. n High specificity to identify,and quantitate metabolites. n Moderate throughput,sample groups are small,n Full validation is required.
Product life stage management and bioequivalence studies. For marketed drugs the application of PK and bioanalysis Assay sensitivity is also important in bioequivalence stud. is predominantly focused on the evaluation of line exten ies as this helps ensure that the elimination phase of the. sion products such as controlled released formulations plasma concentration time curve is defined fully. and on the evaluation of generic formulations, If the lower limit of quantification LOQ is high this can. Relative bioavailability and bioequivalence determina cause problems when estimating the elimination rate. tions are both reliant on quality plasma concentration constant This in turn can cause a problem when calculat. time curve data AUC Cmax and occasionally Tmax are the ing the AUC0 as this is derived using the elimination rate. decision making parameters for the test product constant to estimate the part of the concentration curve. from the time of the last quantifiable concentration to. In a bioequivalence study the aim is to demonstrate. infinity A small CV on assay data will improve accu. that two formulations of an entity have the same, racy in determination of the elimination rate constant and. bioavailability to within certain limits Regulatory. elimination half life,requirements e g U S FDA EMEA guidelines for such. studies are explicit thus the FDA guidance for generic It is important to acknowledge that many factors contrib. products states that the rate and extent of absorption ute to variability in the conduct of bioequivalence studies. are not statistically different for test and reference Inter subject variability is an ever present factor in phar. A GUIDE TO EFFECTIVE METHOD DEVELOPMENT IN BIOANALYSIS Evaluate MS response and develop a multiple reaction monitoring methodology that provides the optimum MS conditions for detection of the compounds of interest Robust chromatography will ensure the analyte s of interest separate from both endogenous interferences and drug metabolites

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