Experiments and Publications

RPLC Gradient Elution of EVAX-005 using Multiple Detections

EVAX-005, developed by iGORi, is a standard mixture of formulation polymers.  The use of EVAX-005 during method development helps to identify coelution of drug substance and formulation excipients.  The standard mixture also helps in extraction optimization.  EVAX-005 is composed of generally used tablet formulation polymers, such as PEG, PVP, HPMC and HPC.

Multiple detections at UV 205, UV 232 and evaporative light scattering (ELS) are capable of visualizing and identifying the components of this complex standard mixture.  Figure 1 display the reversed phase chromatograms of EVAX-005.
Fig. 1 Chromatograms of EVAX-005

Capillary Gel Electrophoresis of Proteins

CGE is a novel version of the traditional SDS-PAGE method for the analysis of proteins and and protein mixtures.  CGE combines the resolution mechanism of gel electrophoresis with the convenience of HPLC instrumentation.  The separation results are displayed in electropherograms, instead of sheets, allowing easy visualization and quantitative evaluation of the separation results.

Protein conformational changes (denaturation) under chromatographic conditions.

Some proteins have very sensitive structure which can be disturbed during purification or analysis.  In most cases the reason for these conformational changes are the interactions of the protein with the chromatographic surface.  The surface and mobile phase induced conformation changes frequently occuring, however one not always recognizes the change.  The various conformations, from a separation point of view, should be considered as new species of the sample mixture and as such they are visualized as new peaks.

Proteins, which are apperently pure, often demostrate odd peak shapes or multiple peaks.  The multiple peak phenomena of pure proteins originates from the kinetics of unforlding-refolding equilibrium.  When the kinetics of conformational equilibrium is much slower or much faster than the time scale of chromatography, only one peak, either the native or the denatured can be observed.  When the conformational equilibrium is on the time scale of chromatography, we can see multiple peaks or odd peak shapes.

Our experience is that this phenomena occurs with most proteins but it is rearly recognized.  However in some cases it could create confusion.  We are studying these types of surface induced conformational changes for over two decades and it is clear that HPLC conditions are excellent to study surface induced conformational events, which are frequently occuring during the formulation of protein and peptide based biopharmaceuticals.

Protein solubility mapping

One of the most important information about a protein is its solubility under different conditions.  Solubility is the key parameter to selecting the best conditions for analytical method development, formulation and production.  Identifying the appropriate solution conditions is mostly done by a trial and error process. 

At iGORi we developed a protein solubility screening system which can assists us to create the solubility map of most biopharmaceutically relevant proteins.  The solubility map is specific for one particular protein.

The iGORi Protein Solubility Map is created from experimental measurements where the pH and ionic strength of the buffers are selected by our optimization.  However, our method is flexible and allows the screening of large numbers of other buffer conditions.

The next Figure displays the solubility maps of two different proteins and clearly illustrates the huge differences in solubility as a function of the proteins.

Fig. 2 Protein Solubility Maps

Solvent effects on extraction

OPADRY  Analytical chemists often face with the problem of poor drug substance recovery during extraction.  This problem can be the results of various phenomena. One of the phenomena involved is the effect of extraction mixtures on the recovery of polymers.  The chemical ingredients of extraction solvents have a significant effect on the composition or structure of the extracted HPMC as analyzed by SEC and displayed in Fig. 2. 

The solvent generated differences of the HPMC composition or structure could also affect the extraction of drug substances.  The drug substance could be entrapped in the polymer network and it is manifested in poor recovery.

EVAX-005 The potential problems during tablet extraction was studied using our EVAX-005, standard mixture. The effects of various solvents on the extraction is displayed in Fig. 3.  The differences are significant as illustrated by the obvious differences in the reversed phase chromatograms.

These results are clearly representing the benefits of an evaluation standard.  The use of EVAX-005 can facilitate the optimization of the extraction step during analytical method development.

Fig. 3 Opadry extraction
Fig. 4 EVAX-005 extraction
Method development tools for the analysis of complex pharmaceutical samples
Kinetics of unfolding of proteins on hydrophobic surfaces of reversed-phase chromatography
Conformational changes of BDNF during RPLC