IEX-HPLC separates molecules based on charge. The protein interacts with the charged moiety on the column and is then eluted with either salt or pH gradients. Elution from the column is from the weakest to the strongest bound. The protein solution is loaded onto a column that has been charged with the counter ion, and is then equilibrated with the starting buffer. Proteins are eluted from the column by a gradient of either salt or pH. If the column with a second buffer contains salt, this disrupts the protein interaction with the column and replaces the protein with the counter ion. If the second buffer changes the pH, this alters the charge on the protein and decreases the interaction of the protein with the column. IEX columns can be either anionic or cationic. The most common anion exchangers are quaternary ammonium, diaminoethyl, and quaternary aminoethyl, and the most common cation exchangers are sulfopropyl, methyl sulphonate, and carboxymethyl. By using buffers above or below the protein's pI, the same protein can be analyzed on both anionic and cationic columns. Because of the ionic nature of the interaction between the protein and the column, the size of the protein does not affect binding. Additionally, because IEX is run in an aqueous environment, the protein is not denatured and maintains its structure, resulting in rendering the method more sensitive to differences such as oxidation in surface amino acids. While this method can be used to assess purity, it is generally less sensitive to purity than RP-HPLC because proteins can remain associated during the separation.

SEC-HPLC is different from RP-HPLC and IEX in two major ways. The first difference is that separation is based on size only, with a small impact from the shape of the molecule. The second difference is that during SEC, the protein does not adsorb or bind to the separation media. In some cases a protein will non-specifically bind to the column. In those instances it is important to use a different column matrix or to change the composition of the running buffer. The molecular weight of a protein is determined by comparing its elution time to the elution time of standard proteins of known molecular weight. Because there is no binding of the protein with the column matrix, the protein separation is sensitive to the sample's volume. After running a set of known proteins through an SEC column, the protein of interest is loaded onto the column in a small volume and eluted under the same conditions. A standard curve is constructed, based on the molecular weight of the standard proteins and elution time. This curve is used to determine the molecular weight of the eluted sample. SEC columns are available that can separate proteins with molecular weights as high as 1,000,000, allowing SEC to be used to identify and quantify the size and amount of aggregates in a protein preparation. Unlike SDS-PAGE, the protein is not denatured before separation, so that non-cross linked aggregates are not disrupted and can be identified. Purity (or percent aggregation) determined by these two methods (SDS-PAGE and SEC) often differs considerably due to the detection of the additional aggregate forms in SEC.

MASS SPECTROSCOPY

The information lost by reducing fragmentation in standard MS can be determined using MS/MS. In MS/MS, specific ions are subjected to an additional energy by collision, and the resulting daughter ions allow even more structural information to be determined, even to the level of amino acid sequence. This technique is especially useful for determining post-translational changes to the protein. MS/MS can also be used to sequence the structure of carbohydrate side chains on glycosylated proteins and to identify the micro-heterogeneity they introduce.