High Performance Liquid Chromatography and Its Applications in Biomolecule Analysis

Date : Aug 23, 2017 Author : Nikhil Kaitwade Category : Chemical and Material

Government authorities are trying to reduce healthcare costs and improve quality with HPLC

In 2015, six of the top 10 bestselling drugs were biomolecules. Unfortunately, five of these are anticipated to lose their patent rights within the next three years, causing a number of biosimilar issues. Thus, the key test will be to fill the potential revenue loss with new biomolecules while also balancing the biosimilar contest as the latter will be lining up for their own revenue stream.

Pharmaceutical companies have typically concentrated on small molecules for drug development, as smaller molecules have lesser impurities in the final product. The transformation to biologics seen in the last three decades is considered to be the future of the Chromatographic Silica Resin Market.

The potential impurities created during the biomolecular manufacturing process can be hundreds of times greater than those with small molecules. Therefore, LC-MS chromatographic methods and analytical techniques should be able to separate complex sample mixtures that are usually found with impurities of a low abundance. The sensitivity challenge has been observed in the analysis of small molecules for several years now and is understood as many solutions have been devised.

In the case of the larger biomolecules, the particular challenges observed are different and it is also compounded by the sheer diversity of molecules. The biomolecule range includes DNA, RNA, Oligonucleotides, monoclonal antibodies, proteins, peptides and amino acids. Some of the most popular chromatographic methods that can be used are ion-exchange, size exclusion, and Ultra-High-Pressure Liquid Chromatography.

Ultra-High-Pressure Liquid Chromatography provides high sensitivity and speed separation but the cost is the backpressure generated and the need for particular instrumentation and the related acquisition costs. When compared to High-Pressure Liquid Chromatography, it can be easily inferred that UHPLC offers greater resolution and sensitivity for the same sample even with a shorter column length. This is mainly due to the small 1.7µm particle used as particle size is inversely proportional to efficiency. Newer core-shell technologies offer high potential and efficiency for shorter analysis times without notable backpressure increases over conventional completely porous particles of a similar size. 

A major limitation of the core-shell technology is loading as roughly 65% of loading capacity can be achieved in comparison to fully loaded particles. A lower surface area of the material has been proven to reduce the quality of the compound injected on the column. While this may not cause much of an issue at the molecule design R&D stage, it could be a problem at the production stage where both loading and resolution can greatly impact the pure drug recovery. Each method has its own particular advantages and disadvantages and the analyst can make a decision of a particle technology to be used by taking all factors into consideration.  

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