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In 2018, the market value of small molecule drug discovery was US$29,363.85 million. The predicted compound annual growth rate (CAGR) of this market until 2024 is 8.11%, says a globally renowned market research firm.

That means the estimated market value of small molecule drugs will reach US$46,882.22 million by 2024. The major driver of market growth for small molecule drugs has been their efficacy in treating chronic diseases.

This article explores the major recommendations for small molecule bioanalysis issued by the FDA.

Major Recommendations For Small Molecule Bioanalysis

There are differences between the processes of small molecule bioanalysis and large molecule bioanalysis. However, the latest Food and Drug Administration (FDA) guidelines issued in May 2018 recommend standards applicable for both. 

The key regulatory requirements are as follows:

1.   Reference Standards And Critical Reagents

It is necessary to characterize and document all reference standards and critical reagents as per their identity, purity, and stability. Critical reagents include, but are not limited to, antibodies, labelled analytes, and matrices. Storing them under defined conditions is also essential.

Certificates of analyses (CoAs) are necessary for commercially available reference standards. Assay validation for any changes in critical reagents need:

  •       Evaluation of binding and reoptimization of assays
  •       Performance verification with a standard curve and QCs
  •       Cross-reactivities assessment

2.   Calibration Curve

The calibration curve of a validated method needs to be continuous and replicable. The detailed requirements for the calibration curve are available on page 20 of the FDA guidelines.

3.   Quality Control (QC) Samples

The FDA recommends fresh QC samples during the method development stage.

4.   Selectivity, Specificity, And Sensitivity

The FDA guidelines recommend at least six analyses of blank samples in the appropriate biological matrix, such as plasma, for CCs. The recommended number for LBAs in 10. This is necessary to determine the selectivity and specificity of the assay.

LLQQ is the recommended method for sensitivity assessment.

5.   Accuracy, Precision, And Recovery

Method validation for accuracy and precision should involve at least three independent runs for CCs and six for LBAs. Each run needs to include a calibration curve and several QC concentrations. It is important to conduct these independent runs over several days.

LBAs do not need recovery experiments. The requirements for recovery experiments of CCs are available on page 20 of the FDA guideline.

6.   Stability And Dilution Effects

The FDA guidelines attach a lot of importance on stability studies, detailing out the following stability parameters:

  •       Autosampler stability
  •       Bench-top stability
  •       Extract or process-sampled stability
  •       Freeze-thaw stability
  •       Long-term stability
  •       Stock-solution stability

The FDA guidelines recommend dilution integrity monitoring during method validation. The dilutions used during validation should be the same as the expected dilutions during the study.

7.   Partial And Cross-Validations

Partial validations are employed to assess changes in bioanalytical methods that have already been validated. It is important to retain raw data of partial validation for inspection purposes.

Cross-validation is necessary to compare the validation methods of two or more bioanalytical methods. It also compares different techniques applied for validation in the same study. If sample analysis happens in more than one site, then also cross-validation is necessary.

In such a situation, each site needs to conduct cross-validation using the same QCs and nonpooled subject samples.

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