Bioanalytical assays validation

Given the foregoing discussion of some of the unique characteristics of macromolecules that lead to clear differences in their pharmacokinetics compared to those typical of small-molecule drugs, there is a subset of the entire group of bioanalytical assay validation parameters that are of key importance in support of pharmacokinetics of candidate macromolecular therapeutics. Assuming demonstration of accuracy and precision of sufficient quality for the intended application of the assay (e.g., non-GLP discovery support or GLP toxicokinetic support, as discussed above), the most important characteristics of a given assay in support of pharmacokinetic studies are likely to be selectivity, specificity, and reproducibility for analysis of incurred samples. These are all related to the ability of the LBA to detect and quantitate solely, or as closely as possible to solely, the analyte of interest. 

It is therefore essential that before pivotal (repeat dose) preclinical studies are initiated, bioanalytical assay development must be completed. This has to cover potential test species, normal and diseased humans. The assays must be validated in the sampling matrix of the toxicity test species, and one should also develop suitable assays for antibodies to the test article. 

Stereoisomer Assays. There are many drugs that are administered as racemic mixtures. They may undergo stereoselective metabolism and/or elimination, and one isomer may be more active than the other. Therefore, there is the need to develop and validate bioanalytical assays for stereoselective determination in bioavailability/bioequivalence studies. All methods used for measurement of stereoisomer should be validated (with emphasis on stereospecificity). For bioequivalence studies of an existing racemic product, a stereospecific assay is not required if the rate and extent of profiles are superimposable (within the usual statistical boundaries) [3,23]. 

Although the lower limit of quantitation is established during assay validation and prior to microdosing, assay sensitivity remains an uncertainty until the actual analysis of the microdose samples as well. There is always the danger that plasma exposures from the microdose are lower than predicted and as a result plasma concentrations from some or all of the time points cannot be detected by the LC-MS/MS method. Reduction of this risk is achieved by collaborative communication between the bioanalytical chemist and the project team. Conservative estimates on bioavailability and clearance can be used to establish the necessary limit of detection needed to determine plasma concentrations for all time points. Updates on the progress of the assay development allow the team to decide if the achievable limit of detection will enable the determination of plasma concentrations from enough time points to make a go-no go decision. Of course, sensitivity is not an issue with AMS, which practically ensures that plasma concentrations will be determined, possibly for several days, enabling the observation of complex PK and clearance from deep compartments. 

Desilva B, Smith W,Weiner R, et at. Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm. Res. (2003) 20 1885-1900. 

Cetuximab serum concentrations were measured using validated enzyme-linked immunosorbent assay (ELISA) methods or a validated surface plasmon resonance assay. These bioanalytical assays were crossvalidated to allow pooling of PK data across studies. 

Viswanathan CT, Bansal S, Booth B, DeStefano AJ, Rose MJ, Sailstad J, Shah VP, Skelly JP, Swann PG, Weiner R (2007) Workshop/conference report — quantitative bioanalytical methods validation and implementation best practices for chromatographic and ligand binding assays. AAPS J 9 E30-E42... 

Viswanathan CT et al (2007) Quantitative bioanalytical methods validation and implementation best practices for chromatographic and ligand binding assays. Pharm Res 24 1962-1973... 

Stability tests in plasma and serum are fixed part in the validation procedures of bioanalytical assays (FDA 2001). 

Hou W, Watters JW, McLeod HL (2004) Simple and rapid docetaxel assay in human plasma by protein precipitation and high-performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography B 804 263-267 Schuhmacher J, Zimmer D, Tesche F, Pickard V (2003) Matrix effects during analysis of plasma samples by electrospray and atmospheric pressure chemical ionization mass spectrometry practical approaches to their elimination. Rapid Communications in Mass Spectrometry 17 1950-1957 Shah PW (2001) Guidance for Industry Bioanalytical Method Validation U.S. Department of Health and Human Services, Food and Drug Administration... 

The performance of the presented bioanalytical assay method was assessed following the guideline for biological method validation presented by Shah et al. (Shah 2001). 

The assay has been validated and the results of validation demonstrate that the standard curve is linear over the concentration range of 100-2000 ng/mL. The assay is reproducible and accurate, with recovery of the analyte and internal standard in the range of 80-90 %. The analysis requires 0.5 mL of plasma and has a limit of quantification of 70 ng/mL. The stability of plasma samples stored at -20 °C has been demonstrated for up to 12 weeks. Autoinjector stability has been demonstrated for over 13 h and freeze-thaw stability has been demonstrated for 3 freeze-thaw cycles. The procedure has a sample throughput of at least 30 specimens per day. The assay meets the guidelines for bioanalytical methods validation for human studies (Shah et al. 1991). 

Validation of bioanalytical assays in general and LBAs in particular has been the subject of intensive debate for the past 18 years or more. Chapter 4 focuses on the key agreements on a phased approach to the validation of LB As, including evaluation of all critical validation parameters prior to implementation of the method to the analysis of any study samples (prestudy validation) as well as in-study validation to assure high performance of the assay during analysis of actual study samples. Also covered in this chapter are the topics of when and how to conduct full validations, partial validations, and cross-validation. 

DeSilva, B., Smith, W., Wiener, R., Kelley, M., Smolec, J., Lee, B., Khan, M., Tacey, R., Hill, H., and Celniker, A. (2003) Recommendations for the bioanalytical method validation of ligand binding assays to support pharmacokinetic assessments of macro molecules. Pharmaceutical Research, 20, 1885 1900. 

Reagents and reference materials Likely will change but should have some documentation on early characterization Continue to screen for optimal reagents Lot no. and history (notebook reference) Evaluate different reagents and identify critical reagents Determine if sufficient quantities are available and their stability for later bioanalytical needs Include C of A for reference materials in assay validation documents Keep records of source and lot no. Use optimized capture/ detection reagents Use characterized reference standard from final manufacturing process with Cof A Record all lot nos. and sources... 

Viswanathan, C.T., Bansal, S., Booth, B., DeStefano, A.J., Rose, M.J., Sailstad, J., Shah, V.P., Skelly, J.P., Swann, P.G., and Weiner, R. (2007) Quantitative bioanalytical methods validation and implementation best practices for chromatographic and ligand binding assays. The AAPS Journal, 9, E30 E42. 

The ultimate goal of an assay or an analytical procedure is to measure accurately a quantity or a concentration of an analyte, or to measure a specific activity, as in some assays for biomarkers. However, many activity assays, such as cell-based and enzyme activity assays, may not be very sensitive, may lack precision, and/or do not include the use of definitive reference standards. Assays based on measurements of physicochemical (such as chromatographic methods) or biochemical (such as ligand-binding assays) attributes of the analyte assume that these quantifiable characteristics are reflective of the quantities, concentration, or biological activity of the analyte. For the purpose of bioanalytical method validation, we will follow the recently proposed classifications for assay data by Lee et al. [4,5]. These classifications, as summarized below, provide a clear distinction with respect to analytical validation practices and requirements. 

Lee, J.W., Nordblom, G.D., Smith, W.C., and Bowsher, R.R. (2003) Validation of bioanalytical assays for novel biomarkers practical recommendations for clinical investi gation of new drug entities. In Bloom J., and Dean R.A. (eds), Biomarkers in Clinical Drug Development. Marcel Dekker, New York, pp. 119 148. 

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