Major Differences between Large and Small Molecule Bioanalysis

Small molecules and large molecules (biotics) differ in properties, behavior, transportation, and more. As such, their bioanalysis requirements also vary drastically.

In drug development, molecules are classified as small and large. Size is a significant difference between the two though, it isn’t the only one. Think about their origin, behavior, transportation, and suitability for different drug forms, the small molecules and large molecules are poles apart. The difference in properties translates into the difference in the types of Bioanalytics involved. Simply put, small molecule bioanalysis differs from the one done for a large molecule.

Small and large molecules:

Typically, small molecule therapeutics (say Aspirin) comprises 20 to 100 atoms. Conversely, the atom-count in large molecule drugs (biotics), say Herceptin range from 5000 to 50,000. While small molecule drugs are often orally administered, biotics typically needs to be injected into the body. Unlike small molecules that interact with cells by penetrating them, biotics works on the cell surface, as their size prevents cell penetration. Small molecules are a result of chemical reactions, whereas, their larger counterparts are manufactured within engineered cells.

Small molecules are better characterized than the large ones. Their purification and bioanalysis can be achieved with routine lab procedures. Biologics, on the other hand, are a potpourri of molecules that might differ from each other, and hence, the difficulty in characterization. The properties of biologics are closely linked to how they are manufactured. Plus, large molecules like proteins reveal a distinctive structural organization pattern, folding. Folding determines how differently two biologics will interact with the body, despite their identical composition.

The difference in bioanalysis:

Biologics are the disruptors in the drug development space. They are well-targeted therapeutics with side-effects restricted to a few overstated pharmacological complications. Even though 90% of drugs are small molecules, the growth in biotics development is 25%. Biologics will account for 30 to 50% of all drugs making their way into the market in the coming decade.

Large molecule bioanalysis requires a new set of techniques, equipment, and approaches. The conventional techniques fail to cope up with the requirements of biotic bioanalysis. We, thus, see the emergence of Ligand binding assays (LBA), MALDI-TOF-MS, HRMS and other cutting edge techniques to facilitate accurate large molecule bioanalysis and expedite drug development.

The biotics’ safety and efficacy need to be established about their ADME properties. Unlike small molecules, biologics induce an immune response from the host body due to their complex structure and high molecular weight. Here, immunogenicity investigations step in. FDA mandates these investigations for large molecule bioanalysis during clinical trials.

Immunogenicity testing is also referred to as Anti-Drug Antibody (ADA) Assays. The body produces antibodies in response to the drug, and the assays identify and characterize the antibodies thus produced. If immunogenicity is determined, regulatory authorities would be interested in gauging the effect on the drug’s activity caused by drug-induced antibodies. That calls for cell-based Nab (neutralizing antibody) assays that resemble the in vitro potency assay.

LC/MS technology is the cornerstone for quick, precise and selective bioanalysis of biotics, as the need for developing antibodies is eliminated outright. Large molecule Bioanalytics requires more in-depth in vitro characterizations at the initial development phases vis-à-vis the small molecule bioanalysis. ESI-MS and MALDI-MS, RP-HPLC–ESI–MS, and other approaches are sought for peptide mapping. Chemical sequencing might also be needed, subject to factors.

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