2.1. Purification of Biologically Active Proteins and Polypeptides
In recent years, a wide variety of important biologically active molecules has been isolated from natural sources or produced by recombinant DNA technology. Many of these molecules are polypeptides or proteins which exert their biological effects by binding to specific cellular receptors.
To purify these polypeptides and proteins, conventional methods such as preparative electrophoresis, gel filtration chromatography, salt or organic solvent fractionation, ion exchange chromatography and the like have often been employed. The problem with the use of such conventional methods is that they are not generally specific for the protein of interest. As a consequence, many of these methods must generally be used in combination before a substantially pure protein can be obtained. Because there are losses due to analytical sampling and incomplete recovery at each purification step, the overall yields are often poor in such multistep purification procedures.
More recently, improved results have been obtained by using immunoaffinity columns containing polyclonal or monoclonal antibodies specific for a protein that is to be purified. For example, Kung, U.S. Pat. No. 4,476,049, has used immobilized monoclonal antibodies specific for the carboxyl terminus of recombinant human immune interferon to purify that protein. The use of immunoaffinity columns can lead to highly purified or even homogeneous protein preparations, but proteins purified in this way may not have their full intrinsic bioactivity.
Pestka, U.S. Pat. No. 4,623,621, has disclosed that many important biologically active polypeptides and proteins can aggregate, forming dimers, trimers and higher oligomeric forms. When such aggregation occurs, the polypeptide or protein may have either lower biological activity or no activity at all. Furthermore, such aggregated material may have the potential for causing deleterious side effects such as antibody production in patients, if it is used therapeutically. Immunoaffinity columns do not necessarily discriminate between the aggregated and unaggregated (monomeric) forms of polypeptides and proteins.
The probability that aggregation of desired polypeptides and proteins will occur is substantially increased when they are produced by recombinant DNA techniques in host organisms and isolated from cell lysates, under conditions and in concentrations that do not occur when they are produced under natural conditions. These conditions can favor aggregation through intermolecular disulfide bridges, other covalent bonds or non-covalent interactions.