The drug development space has focused on developing effective yet safe drug products for decades. Pharmaceutical drugs can be small molecules or large molecules, such as biologics. However, when comparing small vs. large molecules, the proportion of approved large-molecule drugs is smaller. However, this scenario has changed now. Today, more biologics are entering the drug market. Earlier, small molecules had more than 90% of the description market share in the US. But as biologics are developed faster , more large molecule drugs are now entering the clinic and reaching the market shelf.
Experts suggest that large-molecule drugs will comprise almost 50% of the market share. However, large molecules can produce anti-drug antibodies (ADA). ADA immunogenicity testing is crucial for assessing unwanted immune responses. Hence, drug developers are now more focused on analytical techniques for developing and approving biologics and other large-molecule therapeutics.
However, large molecule bioanalysis faces several challenges. Therefore, the current article highlights some challenges in the bioanalysis of large molecules and discusses potential solutions for accurate large-molecule immunogenicity assays assessments.
Challenges and Hurdles in large molecule Bioanalysis
Large-molecule drugs can reach 3000 to 150000 Daltons, compared to small molecules, which are less than 900 Daltons. Hence, bioanalysis of large-molecule drugs is innately complex. Large-molecule protein drugs are more heterogeneous and need robust analytical techniques. They often require multiple sample preparation and separation techniques before performing the actual bioanalysis.
Additionally, large-molecule drugs require multiple characterization initiatives. Some primary characterization approaches include assessing the purity, stability, and function of large-molecule drugs. Researchers usually confirm small molecule structures through higher-resolution methods such as NMR spectroscopy and X-ray crystallography. However, large-molecule drugs require combining high-resolution and low-resolution tools to identify and validate their forms.
Characterizing large molecules requires additional analytical techniques, including reverse phase ion exchange and size exclusion chromatography. Besides, they also need newer methods such as hydrophobic interaction, chromatography, and hydrophilic interaction chromatography. Coupling this additional separation step adds complexity to large molecule bioanalysis and increases the cost and associated time.
Accurate solution for the bioanalysis of large molecule
The robustness, reproducibility, and accuracy of bioanalytical methods used in large molecule bioanalysis are critical for reliable results. Traditional bioanalytical techniques such as LC-MS/MS method validation used in small-molecule bioanalysis are often inappropriate while assessing large-molecule drugs such as recombinant peptides, growth factors, antibodies, and oligonucleotides.
Small molecule drug analysis using LC-MS/MS methods is generally optimized and involves sample preparation steps to enhance assay activity. However, large molecule bioanalysis using LC-MS/MS systems requires a more extensive experimental workflow, including digestion. Today, laboratories employ this indirect analytical method in large-molecule bioanalysis. However, this method is highly laborious and can involve complications such as releasing variable peptides.
Ligand binding assays are also employed during large molecule bioanalysis. However, a recent approach combining ligand binding assay with LC-MS/MS method has addressed multiple bioanalytical challenges. This combination provides the sensitivity and selectivity feature of the LC-MS/MS system and the specificity and dynamic range of ligand binding assay.