Laboratory Testing Blog

Glycosylation is a post-translational modification of proteins that occurs in all eukaryotic cells.  One of the key aspects of characterization of recombinant proteins is their glycosylation.  The oligosaccharides contained in glycoproteins are often responsible for diverse biological functions, such as cellular cytotoxicity and specific receptor binding.  The sugar chains on glycoproteins can mediate biological activity, play a role in receptor-mediated recognition, increase solubility, regulate half-life and exert a stabilizing influence upon conformation.  Specific glycan structures are therefore associated with safety and efficacy attributes of many protein drugs.  Glycosylated and non-glycosylated protein variants often have different functional and pharmacokinetic properties.  Correct glycosyltion is essential if a glycoprotein is to achieve and maintain the structure with full biological activity.  Therefore, quantitative measurement of glycans is important in biopharmaceutical development projects.  The relative amounts of the individual glycan structures are monitored during process development to establish the stability and purification steps during manufacturing.  The same monitoring is required during formulation development and during stability testing.  The glycoform distribution of a protein product is in many cases dependent on the methods and conditions of its production.  It is therefore important to be able to correctly measure the different quantities of non and deglycosylated species within samples of recombinant therapeutic glycoproteins in drug candidate.

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One promising approach for the determination of protein-ligand binding constants is the combination of protein-chip-based technologies with mass spectrometry (MS).  In several publications, researchers have integrated SPR and MS for affinity-based capture and characterization of ligands.  Also discussed is the feasibility of performing Matrix-assisted Laser Desorption/Ionization (MALDI)-MS directly on ligand-bound biosensor surfaces.  A fully in-line system combining an SPR biosensor and electrospray tandem MS has been described.  The SPR platform allows the detection, capture and subsequent digestion and delivery of nanomolar to femtomolar levels of ligand for MS analysis.  SPR/MS is a rapid and powerful approach for identifying binding partners from complex mixtures of components.  Combining this technology with larger microarrays makes it a feasible approach for large-scale ligand-fishing experiments or interaction proteomics analyses.

An alternative approach to label-free determination of protein binding strengths with small molecular ligands is Surface Plasmon Resonance (SPR).  SPR is a method for characterizing macromolecular interactions.  It is an optical technique that uses the evanescent wave phenomenon to measure changes in the refractive index very close to a sensor surface.  The binding between an analyte in solution with a ligand on the sensor surface results in a change in the refractive index.  The interaction is monitored in real time and the amount of bound ligand and rates of association and dissociation can be measured with high precision.  Analysis of kinetics and thermodynamics by SPR can be used to understand the complex mechanisms of molecular recognition events.  The great advantage of SPR is the sensitivity and easy access to kinetic data of the non-covalent binding processes.

The non-covalent binding of small molecules, ligands to proteins, is playing a crucial role in biopharmaceutical research.  This interaction would alter the stereochemistry of a protein molecule (a drug candidate) and also could modify its molecular recognition ability and ultimately its bioactivity.  Therefore, it is important to develop a suitable method which is able to quantitatively determinate the binding strengths of the molecule.  A variety of different approaches have been developed to quantify molecular interactions.  Methods applied to drug-protein binding studies in the pharmaceutical and biomedical sciences include equilibrium dialysis, ultra-filtration, ultra-centrifugation, gel filtration, calorimetry, microdialysis, spectroscopic, HPLC, and capillary electrophoresis-based methods.

Like most analytical CRO's, Diteba relies upon their High Performance Liquid Chromatography (HPLC) systems as being the workhorse of the laboratory.  From assay and purity work on raw materials, to stability sample testing, to bioanalytical analyses, the HPLC instruments are often critical to Diteba's and our sponsor's success.

I would like to highlight some points regarding the application of CE methods in pharmaceutical analysis.  There are three important areas of pharmaceutical analysis where capillary electrophoresis (CE) can be successfully used: