Reproducibility bioanalytical

Bioanalytical laboratories provide support for most of the activities at the biopharmaceutical company. They are responsible for characterizing the molecules in development, establishing and performing assays that aid in the optimization and reproducibility of the purification schemes, and optimizing the conditions for fermentation or cell culture, including product yields. Some of the characterization techniques will eventually be used in quality control to establish the purity, potency, and identity of the final formulation. 

I also wish to thank Dr Lou Coury and Dr Adrian Bott of Bioanalytical Systems, Inc. for their enthusiasm, and permission to reproduce Figures 6.16, 6.18, 6.19, 10.1 and 10.3.1 gladly thank Dr Manfred Rudolph for his description of the DigiSim program. Dr Mike Dawson of E G G for his help concerning the Condecon program, and Dr Keith Dawes of Windsor Scientific for his help, and the permission to reproduce Figure 10.2. 

As with the determination of the intralaboratory repeatability, the intralaboratory reproducibility was determined by analyzing a cleaned sediment extract and a 3-pM 2,3,7,8TCDD standard on 10 separate days and by multiple persons. The reproducibility for the 3-pM 2,3,7,8-TCDD standard was found to be 13.8%, whereas the reproducibility for the cleaned sediment extraet was shown to be 19.9%. Since the observed reproducibilities are in the range of relative standard deviations for two sediment extracts analyzed in 10-fold on the same day (intralaboratory repeatability), the DR CALUX bioassay can be evaluated as a stable and robust bioanalytical tool. 

The fundamental parameters for bioanalytical validations include accuracy, precision, selectivity, sensitivity, reproducibility, stability of the drug in the matrix under study storage conditions, range, recovery, and response function (see Section 8.2.1). These parameters are also applicable to microbiological and ligand-binding assays. However, these assays possess some unique characteristics that should be considered during method validation, such as selectivity and quantification issues. 

Figure 6.3 Schematic of an ultrafiltration probe manufactured by BASi. Figure reproduced with permission. Copyright BASi. www.bioanalytical.com.

Figure 3.2 The divided electrochemical cell for electrosynthesis. (From Bioanalytical Systems, A Handbook of Electroanalytical Products, Bioanalytical Systems, Inc., reproduced with permission.)...

The authors are very grateful to Bioanalytical Systems, Inc. and Professor Richard G. Compton (Oxford University) for permission to reproduce the schemes of electrochemical cells and to Professor Ubaldo Ortiz Mendez (Universidad Autonoma de Nuevo Leon, Monterrey, Mexico), Professor Igor V. Melikhov, and Professor Sergey S. Berdonosov (both from Moscow State University, Russia) for useful suggestions and comments in preparation of this section, as well as to Professors Takeko Matumura (Japan), Christopher R. Strauss (Australia), and Martine Poux (France) for permission to reproduce the schemes of microwave equipment. 

Bio analytical Methodology Bioanalytical methods for BA and BE studies should be accurate, precise, selective, sensitive, and reproducible. A separate FDA guidance entitled Bioanalytical Method Validation (May 2001) is available to assist sponsors in validating bioanalytical methods. 

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). 

The signal in thermospray LC/MS is the result of an interweaving play of many interdependent experimental parameters. Systematic studies have been undertaken in order to optimize the sensitivity and the reproducibility of thermospray LC/MS for bioanalytical applications (3). During and as a result of these systematic studies optimization strategies for thermospray LC/MS have been developed. 

Repeatability is obtained when the analysis is performed in one laboratory by one analyst using the same equipment at the same day. Repeatability should be tested by the analysis of a minimum of five determinations at three different concentrations (low, medium and high) in the range of expected concentrations, according to FDA [16], However according to the ICH [79] repeatability could be measured by the analysis of three determinations at three different concentrations or through six determinations at 100 % of the test concentration. The latter one is for analysis when the concentration is supposed to be constant for all samples, e.g., pharmaceutical products. The acceptance criteria for precision depends much on the type of analysis. For compound analysis in pharmaceutical quality control, precision should be better than 2 % [82], For bioanalytical applications the precision values at each concentration level should be better than 15 % except for the lower limit of quantification (LLOQ) where is should not exceed 20 % [16], The intermediate precision shows the variations affected in day-to-day analysis, by different analysts, different instruments etc. Reproducibility, as above, represents the precision obtained between different laboratories. 

Applicable principles of GLP should be followed in the conduct of chemical analysis (5). Bioanalytical methods should meet the requirements of specificity, sensitivity, accuracy, precision and reproducibility. Knowledge of the stability of the API and/or its biotransformation product in the sample material is a prerequisite for obtaining reliable results. 

Figure 13.1 Analysis of catecholamines in urine (reproduced with permission of Bioanalytical Systems). Conditions stationary phase, octylsilane mobile phase, citrate-phosphate buffer (pH 4) containing 7% methanol and SOrngl" sodium octyl sulfate electrochemical detector, +700 mV for sample preparation see R.M. Riggin et a ., Anal. Chem., 49, 2109 (1977). Peaks (with concentrations in urine) 1 = norepinephrine (160ngml ) 2 = epinenphrine (31ngml ) 3 = dopamine (202ngmr ) IS —3,4-dihydroxybenzylamine (internal standard).

QCs are prepared at a central laboratory and sent to various clinical chemistry laboratories that participate in the program. The mean observed values among the laboratories, not the theoretical values of the QCs, are used for laboratory proficiency evaluation. Criteria are set differently for different tests by CLIA 88 regulations. Also, bioanalytical laboratories involved in a drug-development program may exchange QCs and test samples to ensure lab-to-lab reproducibility in the analyses. 

An additional bioanalytical parameter of great current interest [54] is reproducibility of analysis of samples from dosed subjects (the so-called incurred samples). Whether such analyses are to be conducted in all species or most appropriately... 

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. 

The ideal PK immunoassay standard curve, which is nonlinear and heteroscedastic, is derived from solutions of well-characterized macromolecules added to a relatively nonreactive sample matrix. However, rarely does one encounter an ideal situation when describing bioanalytical methodology, thus developing and validating analytical methods for macromolecules, and analyzing samples from preclinical or clinical trials must include an evaluation of these variables and possibly many others. Careful consideration must be given to the topics described in this chapter to achieve the goal of accurate and reproducible quantification of biotherapeutics necessary for pharmacokinetic analysis. 

Very little regulatory documentation directly relates to method transfer and usually where it is mentioned, it refers to cross-validation of the method between two laboratories. It is mentioned in the FDA s Bioanalytical Methods Validation Guidance [1] page 3 under Cross-Validation, When sample analyses within a single study are conducted at more than one site or more than one laboratory, cross-validation with spiked matrix standards and subject samples should be conducted at each site or laboratory to establish inter-laboratory reliability. It is also mentioned in the Appendix of the same guidance that defines reproducibility as the precision between two laboratories. ... 

In addition to analytical and nonanalytical repeats, some samples in certain studies need to be reassayed to demonstrate incurred sample reproducibility (ISR). This type of testing is a critical step for demonstrating the reproducibility of the bioanalytical method with samples from dosed subjects (as distinct from precision demonstrated with QCs, i.e., blank matrix spiked with drug). Written procedures in this regard should include a description of how the samples are selected for reanalysis, the comparison and reporting of the original and repeat results, and the acceptance criteria for variability between results. 

The repeat results from this type of testing are used to confirm the reproducibility of the assay and should be presented in the bioanalytical study report as a separate data table. In cases where repeat testing of incurred samples does not confirm that the method is reproducible, the bioanalytical laboratory should investigate the cause of the nonreproducibility before continuing its use of the method or reporting results from samples previously assayed. 

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