The important role of chromatography in the lab

Chromatography has taken a prominent place in the characterization and analysis of protein therapeutic drugs and today it plays a critical role in the biotechnology laboratory. Although reversed-phase chromatography is the foremost chromatography technique used for this purpose, other techniques such as ion-exchange, size-exclusion, normal-phase, hydrophilic-interaction, and hydrophobic-interaction chromatography play very specific roles in characterizing and analyzing protein drugs.

Chromatography has long been an essential tool for protein purification. Dextran-based ion-exchange and size-exclusion columns have been instrumental in protein purification and characterization for decades. With the development of recombinant DNA proteins for therapeutic purposes there has been a need for careful, detailed characterization of these proteins, the changes and modifications that might occur, as well as sensitive determination of changes that actually occur. Chromatography has taken its place as a premier technology in both the characterization and the analysis of recombinant protein therapeutic drugs (1).

Protein Modifications and Their Effect on Protein Therapeutic Drugs

The biological activity and, therefore, the therapeutic efficacy of protein drugs depend on the exact composition and three-dimensional structure of the protein. Any change to a protein (modification) may affect its efficacy as a therapeutic agent, although efficacy may not be affected by some protein modifications. The effect of a given modification depends on the protein and the nature and location of the modification. During development of a protein therapeutic drug it is necessary to fully characterize the protein, define what possible modifications may occur, define those modifications that do affect efficacy (critical quality attributes), and develop methods to analyze such modifications. Changes occur to proteins from the beginning of their assembly. Some changes may be needed for the protein to be properly formed into an active tertiary structure such as glycosylation and disulfide bonds, which are critical in maintaining the proper structure. Glycosylation attaches sugar chains to either asparagine or serine residues. These sugar chains affect protein folding and assist in maintaining correct tertiary protein structure. Glycosylation structure is complicated and is affected by the cell lines used for protein expression as well as other aspects of the expression system. It is vital to characterize and monitor glycosylation patterns. Disulfide bonds formed between cysteine residues are essential for formation and maintenance of proper tertiary structure. These must be characterized as to their locations in the primary structure of a protein and also must be monitored to ensure that a protein remains active.

Other modifications may affect protein binding and reduce activity such as chemical deamidation — the conversion of an asparagine residue into an aspartic or isoaspartic acid residue under conditions of high temperature or high pH — and oxidation of methionine residues under chemical oxidative conditions to methione sulfoxide. These two modifications may affect activity if found near catalytic or binding sites, but may have no effect if found distant from sites of activity.

Some modifications are engineered into proteins to form more effective drugs. The addition of polyethylene glycol to proteins — pegylation — usually extends the duration of protein activity in the blood stream several times by reducing the rate at which the protein is removed from the blood stream. Monoclonal antibodies (mAbs) that bind to specific receptors on the surface of tumor cells can be modified by the addition of highly toxic drugs to the monoclonal antibody, creating a highly specific, effective reagent that targets and kills specific tumor cells. Engineered modifications must be analyzed and monitored.

Proteins may lose tertiary structure by variations in pH, temperature, or the presence of certain reagents. Such “denatured” proteins lose biological activity and pharmacological potency. Proteins may also form “aggregates,” where multiple proteins self-associate into larger complexes that usually reduce activity and may engender immune responses in patients.