Biopharmaceutical Characterization Techniques for Early Phase Development of Proteins - - BioPharm International


Biopharmaceutical Characterization Techniques for Early Phase Development of Proteins

BioPharm International

An electrophoretic method that is being used increasingly in early-stage characterization of proteins is two-dimensional electrophoresis (2-D). This method separates the proteins in one dimension based solely on charge (IEF), and in the second dimension by size (SDS-PAGE). This powerful method can determine whether a protein that is a single band on SDS-PAGE is co-migrating with another protein. A new use of 2-D electrophoresis is for determination of host-cell proteins. This method can often identify host-cell proteins which co-migrate with the protein of interest in SDS-PAGE gels. The protein is separated in a thin IEF gel, the lane is then placed across the top of an SDS-PAGE gel, and a second electrophoresis is run. After staining, the gel contains one or more spots. The gel can be scanned on a densitometer, and the relative intensity of each spot can be used to determine the percentage of protein that is not product.


High performance liquid chromatography (HPLC) is a core technique in characterization of proteins. HPLC separations are coupled with detectors that are sensitive to the proteins eluted during chromatographic separation. The most common detector used in HPLC measures the UV absorption of the eluate at one or more wavelengths or, in the case of a diode array detector, it can scan all the wavelengths simultaneously and provide a clear quantification of each separated protein. Other detection methods sometimes used with HPLC separations are evaporative light scattering and refractive index. The three most common types of HPLC are size exclusion chromatography (SEC), which separates based on the size or molecular weight of the protein; ion exchange chromatography (IEX), which separates based on the charge of the protein; and reverse phase chromatography (RP), which separates based on the hydrophobicity of the protein. RP is such a common HPLC method that when people do not specify a particular HPLC method, they usually are referring to RP-HPLC.

In RP-HPLC, separation of proteins is accomplished by differential interaction with the column matrix and the column buffer. Two buffers, called the aqueous buffer and the organic buffer (thus identifying the most important attribute of each) are used, and the separation is done with a gradient of these buffers. The most common organic buffers are based on acetonitrile, though other organic solvents such as methanol or tetrahydrofuran may be used. The column used for the RP-HPLC separation is most commonly a silica base, coated with hydrocarbon chains of varying sizes, such as C4, C8, and C18. RP columns built on polymer backbones are becoming more readily available. To minimize any nonspecific interaction between the protein and the column matrix, an ion-pairing component, frequently trifluoroacetic acid, is added to both the aqueous and organic buffers. After the column is equilibrated with either the aqueous buffer or a defined mixture of the aqueous and organic buffers, the sample is loaded onto the column as an aqueous solution. Separation of the varying proteins is done by running a gradient of increasing organic buffer; proteins are resolubilized when the hydrophobic nature of the particular protein partitions into the buffer. The use of very hydrophobic buffers for RP-HPLC usually precludes the presence of large amounts of salt, which destabilizes some proteins. Additionally, proteins are denatured in RP-HPLC, so tertiary and quaternary structure is lost. The subunits of multi-subunit proteins will usually elute separately. Multiple forms of a protein can usually be separated in RP-HPLC by their small differences of hydrophobicity and sometimes molecular weight. RP-HPLC is often considered to be a good method to separate related isoforms of a protein.

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