Optimization of protein G chromatography for biopharmaceutical monoclonal antibodies.

Compared to more stable chromatographic media the use of affinity media with biological ligands such as Protein G Sepharose 4 Fast Flow poses special challenges regarding regeneration and sanitization. This is especially critical for the purification of pharmaceutical proteins, where complete regeneration of the column between runs is of paramount importance. Here, the problems encountered during process development and upscaling of regeneration methods for a Protein G Sepharose Fast Flow column intended for the large-scale purification of pharmaceutical monoclonal antibodies are reported. The initially chosen alkaline regeneration buffer led to an increase in the affinity of Protein G towards antibodies which made elution increasingly difficult. A combination of urea and acetic acid was selected to ensure efficient cleaning of the matrix without affecting ligand properties. Validation experiments were done to demonstrate the functional integrity of the matrix after repeated cycles of use and regeneration, as well as the efficiency of the cleaning process.

Separation of fibronectin from a plasma gelatinase using immobilized metal affinity chromatography.

Conventional preparations of plasma fibronectin are known to contain a co-purifying gelatinase [1986, J. Biol. Chem. 261, 4363-4366], but so far useful methods to remove the protease have not been available. In this study a number of different methods were tested in order to achieve separation of the two proteins. Immobilized metal affinity chromatography was found to be efficient for this purpose, and a convenient procedure to separate the two proteins under nondenaturing conditions on chelating Sepharose charged with Co2+, Ni2+, or Zn2+ is described. An alternative method employing pH gradient elution of an Fe3+ gel also resolved fibronectin from the gelatinase. The Fe3+ gel bound both proteins at pH 6.0 but not at pH 7.4, suggesting that the two proteins were phosphorylated. The described procedures will now allow studies of the functions of fibronectin in the absence of the contaminating protease.

Purification of membrane proteins in SDS and subsequent renaturation.

A prerequisite for the purification of any protein to homogeneity is that the protein is not non-specifically associated with other proteins especially during the final stage(s) of the fractionation procedure. This requirement is not so often fulfilled when nonionic detergents (for instance Triton X-100) are used for solubilization of membrane proteins. The reason is that these detergents are not efficient enough to prevent the protein of interest from forming aggregates with other proteins upon contact with chromatographic or electrophoretic supporting media, which, due to their polymeric nature, have a tendency to induce aggregation of other polymers, for instance, hydrophobic proteins. The aggregation can be avoided if sodium dodecyl sulfate (SDS) is employed as detergent. We therefore suggest that membrane proteins should be purified by conventional methods in the presence of SDS and that the purified proteins, which are in a denatured state, are allowed to renature. There is good change to renature internal membrane proteins since they should not be so susceptible to denaturation by detergents as are water-soluble proteins because the natural milieu of the former proteins is lipids which in fact are detergents. In this paper we present a renaturation method based on the removal of SDS by addition of a large excess of G 3707, a nonionic detergent. By this technique we have renatured a 5'-nucleotidase from Acholeplasma laidlawii and a neuraminidase from influenza virus. The enzyme activities were higher (up to 6-fold) after the removal of SDS than prior to the addition of SDS.