Drug development is a complex process, warranting extensive testing of the lead therapeutic candidates. Regulatory guidance mandates analytical tests throughout the preclinical and clinical stages of drug development. This helps to judge the suitability of the lead therapeutic candidate to be developed and launched as a drug in the market. Like small molecule bioanalysis, large molecule bioanalysis solutions include several bioanalytical tests.
What is Large Molecule Bioanalysis
Large Molecule Bioanalysis falls within the scope of analytical chemistry in which large molecules with therapeutic prospects, such as proteins and monoclonal antibodies, are analyzed within a matrix such as serum or plasma. Large Molecule Bioanalysis includes diverse tests, including pharmacokinetic, toxicokinetic, pharmacodynamic, and immunogenicity bioanalysis. In addition, large molecule bioanalysis includes bioanalytical tests for biomarker discovery and validation. These bioanalytical tests are performed using different types of high throughput assays, and in some cases, mass spectrometry services are employed for large molecule bioanalysis.Â
Best Practices Involved
Best practices in large molecule bioanalysis encompass aspects of method development, method validation, sample preparation, data acquisition, data analysis, and data reporting. The adherence to best practices ensures the quality, reliability, and integrity of results obtained from bioanalytical studies.Â
US FDA encourages the development of new bioanalytical techniques for sample analysis. The new technique requires proper validation to ensure that the dataset generated using the new method agrees well with the results obtained from other validated analytical methods. Including two analysts during method validation is a widely accepted best practice. In addition, the US FDA encourages the comparative assessment of the output of the new method with another validated method using a set of a minimum of 20 incurred samples.
Sample preparation is a crucial aspect of large molecule bioanalysis involving matrix. Care must be taken to optimize the method, minimize potential method-related problems, and include options to identify problems. When conducting analysis, an authentic matrix should be used to prepare the standard or quality control sample to normalize matrix effects. Reagents must be brought to room temperature before using them. Researchers are cautioned against repeated freeze-thaws for large biomolecules to avoid getting denatured. Instead, it is best to aliquot them into small volumes and freeze them. Using multichannel pipettes for assays can help in achieving more consistent results.
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The use of internal standards for liquid chromatography is a well-accepted best practice. Using 2 to 3 internal standards with different retention times while analyzing samples with differing retention times is recommended. A marker compound can also be included to indicate data quality. Including single or double-blank samples containing only internal standards within the matrix can help to observe matrix interference, if any.
Using a standard is a best practice that applies to other bioanalytical tests, including assay-based methods. Analyzing samples in replicates and using positive controls and blank samples is recommended. It is crucial to dilute test samples so that their concentration falls within the linear range of the standard curve.
Within the scope of data acquisition, best practices include optimized methods and regular calibration of instruments involved in bioanalytical studies. In data acquisition using LC-MS, it is beneficial to include two mass transitions as a precautionary measure if the matrix effect interferes with one of the transitions.
Once the acquired data is analyzed, the investigators should report all the relevant information about the experiments conducted to the concerned authorities, including the repetitions and conditions of sample analysis.Â
Conclusion
Drug development is inherently complex and more so for large molecules and biosimilars, and adherence to best practices in the bioanalysis of such molecules ensures the quality, reliability, and integrity of results. Further efforts should aim to raise the existing bar of best practices.
