Recommendations on qPCR/ddPCR assay validation by GCC.

Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.

Recommendations on ELISpot assay validation by the GCC.

Gene therapy, cell therapy and vaccine research have led to an increased need to perform cellular immunity testing in a regulated environment to ensure the safety and efficacy of these treatments. The most common method for the measurement of cellular immunity has been Enzyme-Linked Immunospot assays. However, there is a lack of regulatory guidance available discussing the recommendations for developing and validating these types of assays. Hence, the Global CRO Council has issued this white paper to provide a consensus on the different validation parameters required to support Enzyme-Linked Immunospot assays and a harmonized and consistent approach to Enzyme-Linked Immunospot validation among contract research organizations.

The effect of AST-120 on the single-dose pharmacokinetics of losartan and losartan acid (E-3174) in healthy subjects.

AST-120 is an orally administered adsorbent used to slow the progression of chronic kidney disease (CKD). This was a randomized, open-label, 5-way crossover study to assess the effect of AST-120 on the pharmacokinetics of losartan and its active metabolite (E-3174) in healthy subjects. Losartan (100 mg) was administered alone under fasting (A) and fed (B) conditions, and results were compared when AST-120 (3 g thrice daily for 2 days) was administered 60 minutes after (C), 30 minutes prior to (D), and 30 minutes after (E) losartan. Plasma concentrations of losartan and E-3174 were assayed by high-performance liquid chromatography with mass spectrometry detection. Under fed conditions, treatment C had no significant effect on the AUC(0-t) and Cmax of losartan and E-3174. Treatments D and E resulted in a marked decrease in Cmax of losartan and E-3174. Therefore, administration of AST-120 60 minutes after losartan under fed conditions may be preferred over other dosing regimens for CKD patients.

Validation study of a method for assaying DE-310, a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, and the free drug in human plasma by HPLC and LC/MS/MS.

DE-310 is a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, DX-8951, conjugated to a water-soluble polymer by means of a peptide spacer. New assay methods have been developed to determine the polymer-bonded DX-8951 conjugate, free DX-8951, and Glycyl-DX-8951 in human plasma. Solid-phase extraction was used to extract free DX-8951 and Glycyl-DX-8951 from plasma, and LC/MS/MS (Method I) was used to determine the amount of each analyte. Protein precipitation was used to extract Conjugated DX-8951, which was then digested with thermolysin. HPLC (Method II) was used to determine the productive compound (Phenylalanyl-Glycyl-DX-8951). The lower limit of quantitation of DX-8951 was 50 pg/ml, of Glycyl-DX-8951 was 80 pg/ml, and of Conjugated DX-8951 was 100 ng/ml (as DX-8951 equivalent). Both methods showed satisfactory sensitivity, precision, and accuracy.

Testing equivalence between two laboratories or two methods using paired-sample analysis and interval hypothesis testing.

A modified interval hypothesis testing procedure based on paired-sample analysis is described, as well as its application in testing equivalence between two bioanalytical laboratories or two methods. This testing procedure has the advantage of reducing the risk of wrongly concluding equivalence when in fact two laboratories or two methods are not equivalent. The advantage of using paired-sample analysis is that the test is less confounded by the intersample variability than unpaired-sample analysis when incurred biological samples with a wide range of concentrations are included in the experiments. Practical aspects including experimental design, sample size calculation and power estimation are also discussed through examples.