Bioanalysis of large molecules implies qualitative and quantitative analysis of biological drugs or biologics. They may include gene therapies, tissue transplants, monoclonal and polyclonal antibodies, drug-antibody conjugates, stem cell therapies, and recombinant proteins used as therapeutics.
Bioanalysis of Large Molecules
Analysis of large biomolecules that are potential drug candidates helps to evaluate their pharmacokinetics, pharmacodynamics, efficacy, and safety when formulated as a therapy. In contrast to small molecules, large biomolecules have intricate structures that give rise to distinct pharmacokinetic properties, and analyzing these properties can help to understand how the biological molecule of interest would behave inside the body in terms of their absorption, distribution, metabolism, and excretion (ADME), and this in turn, helps to assess the safety of the drug and optimize its dosage.
Complex diseases such as genetic and autoimmune diseases are often beyond the therapeutic scope of small molecules. The lack of effective therapeutics and the need for better treatment outcomes has accelerated the development of biological drugs. Currently, biologics is a growing market that is set to expand rapidly in the future. With the increased demand for biologics, the need for bioanalysis of large molecules has escalated.
Leveraging this demand, clinical research companies and other biotech organizations have started offering clinical bioanalysis services for small and large molecules. These help to accelerate the development of biologics in pharmaceutical companies. Clinical bioanalysis services for large molecules include biomarker discovery and validation, pharmacokinetic studies, pharmacodynamic studies, toxicokinetic studies, and immunogenicity testing such as immunogenicity ADA and NAb assays.
Techniques and Challenges
Techniques commonly employed for the bioanalysis of large molecules include immunoassays such as ELISA and ELISpot, ligand binding and cell-based assays, ADA and NAb assays, plaque reduction neutralization test (PRNT), chemiluminescence, surface plasmon resonance (SPR), flow cytometry, and mass spectrometric methods such as LC-MS/MS.
The bioanalytical methods need to be developed and subsequently validated to ensure they yield consistent and reliable results. Biologics development is governed by regulatory guidelines outlining the criteria to be adhered to while validating, analyzing, and reporting data. Therefore, method development and validation must strictly obey the regulatory guidelines to ensure accuracy, precision, sensitivity, specificity, and reproducibility.
Unlike small molecules, large biomolecules have complex structures and large sizes. Biologics have inherent risks of immunogenicity since the patient may produce antibodies against the drug, adding to the challenge of developing biologics.
Analysis of large biomolecules requires highly sensitive and specific analytical techniques, and each method has its technical limitations. For example, immunoassays are prone to drug interference; consequently, the binding of ADA and NAb can simply go undetected or underestimated.
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ELISA, for example, has a complex workflow and protocols, which limits its potential for high-throughput screening and analysis, which would be extremely time-consuming. Furthermore, traditional ELISA generates a large amount of data, thus creating bottlenecks for data analysis. Powerful analysis software can help to mitigate the challenges of time-consuming data analysis.
Flow cytometry is time-consuming mainly due to the slow process of cell sorting. Cell viability reduces during the analysis, so one must begin analyzing with large amounts of cells. Cell cytometry can analyze only those cells suspended in a culture and not adhered to a medium. It also fails to analyze cell-cell interactions.
On the other hand, the mass spectrometric method also has disadvantages. It cannot distinguish optical and geometrical isomers and compounds having similar fragmentation patterns. Furthermore, polar biological molecules often have poor ionization efficiency.
It is possible to overcome these technical challenges using advanced technologies, combining multiple techniques, and expanding our knowledge domain.
