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A non-denaturing high-performance size-exclusion chromatographic method has been developed for the determination of the potency of recombinant bovine somatotropin (rbST) in bulk materials. A Spherogel TSK 3000 PW column containing a polymer base packing material with very hydrophilic bonded surface was used in this method. Ammonium hydrogencarbonate buffer pH 9.0, was used as the mobile phase. This method was shown to be non-denaturing by rat mass-gain assay and radioreceptor assay. The optimization for the separation and determination of rbST has been investigated. The method was validated for the determination of rbST in bulk materials. In addition, rbST soluble aggregates formed in the production process due to protein association, used to be found in bulk materials. The behavior of rbST soluble aggregates in ammonium hydrogencarbonate solutions have been studied. The bio-inactive aggregates can be separated by the method developed in this study. The high-low chromatographic technique has been used to estimate rbST soluble aggregates in bulk materials.

Column plate numbers, N, were measured for 12 different proteins as a function of mobile phase flow-rate in two gel filtration systems (either denaturing or non-denaturing conditions). These data were used to extend a previous model that predicts bandwidths in reversed-phase and ion-exchange chromatography. Restriction of diffusion of large molecules within column packing pores is now defined more precisely, with a single relationship describing this effect for both reversed-phase and size-exclusion chromatography (SEC) (and presumably other high-performance liquid chromatography systems). Separations by gel filtration (SEC) are now included in our general model. A total of 17 flow-rate studies were carried out, involving different proteins, columns and/or mobile phase conditions (denaturing or non-denaturing). Comparisons of plate numbers predicted by the model with experimental values were satisfactory in 15 out of 17 cases. The remaining two cases appear to represent "non-well-behaved" systems, where experimental bandwidths were higher than predicted values by more than 20%. Initial attempts at understanding the origin of these non-ideal effects are described.

A general model for describing gradient elution separations of peptides and proteins by reversed-phase high-performance liquid chromatography (HPLC) has been presented previously. This model has now been modified so that it can be applied to any of the four HPLC methods used for separating biological macromolecules: reversed-phase, ion-exchange, hydrophobic-interaction and size-exclusion chromatography, carried out in either an isocratic or gradient elution mode. The role of sample molecule structure and the particular column used has been further studied, so that previous empirical parameters for different column/sample choices can now be estimated from three physical properties of the sample and the column: sample molecular weight, native vs. denatured sample, column packing pore diameter. This eliminates much of the empiricism of our preceding model, and minimizes the number of experimental runs now required in order to apply the model in practice. The final model has been tested for several hundred runs involving peptides and proteins in the molecular weight range 600-162,000, all four of these HPLC methods, in both isocratic and gradient elution modes, and using data from several different laboratories (including our own). The model is able to predict bandwidth in HPLC separations of proteins and peptides with an accuracy of +/- 17% (1 standard deviation), for the case of "well-behaved" separations. Separations that are not "well-behaved" will give wider bands than predicted by the model.