38
May/June 2013
Optimising Protein and Peptide Speed and Resolution with Superficially Porous Columns
by Jim Martosella, Phu Duong and Alex Zhu Agilent Technologies, Wilmington, DE 19808, USA
Proteins and peptides are large biological molecules consisting of a chain of amino acids. They have a wide variety of functions within a physiological system, ranging from structure formation, cell signalling, enzymatic to transportation processes, with the only difference between the different molecules being the order and number of the backbone amino acid structure. Since in a biological entity there are only a finite number of amino acids this can result in structures that are very similar. Both of these types of compounds can be highly hydrophilic in nature and so to achieve a retention in a chromatographic environment, a suitable chemistry has to be chosen. The size of these types of molecules is something that also has to be considered, since their effective diffusion rates are very slow compared to the much smaller molecules that were traditionally analysed within the pharmaceutical industry. The low diffusion rate affects the physical dispersion processes that occur within a column and these have to be carefully considered when designing the stationary phase, to ensure that mass transfer effects are minimised. In particular the pore structure has to be carefully designed to ensure that surface area is maintained but that minimal dispersion due to mass transfer effects occurs.
Column Technology to Aid Speed Advances
The use of superficially porous materials can reduce the effect of mass transfer effects, by reducing the pore depth [1,2]; however it is important that the pore diameter is large enough to cope with the analysis of these larger molecules. The column technology to aid the separation scientist to achieve these goals has been developed and in particular the use of superficially porous columns with, 300 Å pore diameters for proteins and 120 Å pore diameters for peptides, addresses the challenges associated the mass transfer effects. The work presented here demonstrates how the use of the correct column chemistry coupled with the correct pore size can ensure that fast high-resolution separations of protein and peptides are obtained. Optimisation of the separation also provides increased resolving power for peptide mapping.
Another advantage that is provided by the use of superficially porous materials is the low impedance, which means that the highly efficient chromatography can be obtained without the need to go to ultra high pressure chromatographic systems [3]. To illustrate the impact of this technology, optimised chromatographic methods compatible with standard 400 bar HPLC system pressures have been developed to enable the rapid profiling of IgG1, degraded Insulin and EPO protein.
Figure 1: Examples of superficially porous column technology Materials and Methods
The samples that were analysed were IgG1 which was used to highlight the separation of a protein and its impurities, and an enzyme digested recombinant human EPO as an example of the peptide separation, both purchased from Creative Biolab, Shirley, NY.
EPO Digestion Procedure
Trypsin protease was added to a solution containing approximately 4.2 mg (2.1 mg/ml, 2 ml) EPO. The ratio of substrate and enzyme was 50:1(w:w). The mixed solution was incubated at 37o
quenched by storing the sample at -70o
C for 12h. The digestion was C.
After BCA analysis, 3.78 mg (2.1 mg/ml, 1.8 ml) of digested EPO was obtained.
Instrumentation
Intact and fragmented Protein Analysis: Agilent 1200 and Agilent Infinity 1290 Infinity LC system with auto injector (HiP-ALS), binary pump, thermostatted oven (TCC) and diode array detector (DAD).
Peptide Mapping: Agilent Infinity 1260 Bio- IgG1 Papain Digestion
To prepare the FC and Fab fragments, a Pierce Fab Micro Preparation Kit was used. The final Fab and Fc clean-up used microcentrifugation with a spin column to separate from the immobilised papain. The final concentration of the fragments was 2 µL / µg.
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