Recombinant human insulin-11. Size-exclusion HPLC of biotechnological precursors. Factors influencing on retention and selectivity

The mechanism of exclusion-sorption interaction of insulin-containing proteins with the column support in accordance with the chemical and three-dimensional structure in native and denaturating conditions was observed. The mechanisms of weak adsorption of linear proinsulin and fusion protein presenting in native, and dynamics of formation SDS-protein complex in denaturating conditions have been revealed. The obtained results are used for SE HPLC analysis of the main and intermediate products practically at all steps of recombinant human insulin production technology. Size-exclusion or gel-permeation liquid chromatography is one of the types of chromatography, in which separation occurs, as the result of the fact, that the specimen molecules penetrate into sorbent pores, filled with the solvent, and are retained there for different time. Molecules, having a larger size in solution, either don't penetrate at all or penetrate only into part of the gel pores and are washed out of the column earlier, than small molecules, as the results of which separation according to the size that specimen molecules having in solution, is ensured (ref. 1). SE HPLC is often used for qualitative and quantitative analysis of proteins and peptides both in wide (ref. 2,3) and narrow (ref. 41 ranges of molecular masses, and can be performed both in native (ref. 5 ) and denaturating (ref. 6,7) conditions. In performing analysis by means of SE HPLC, for qualitative separation of proteins, it is necessary to take into consideration many factors,such as concentration and viscosity of the applied specimen (ref. 1,8) , the choice of geometric (ref. 3 ) and physical-chemical (ref. 9,lO) parameters of column support, elution conditions within different pH limits (ref. 111, temperature (ref. 12), the value of the solution ion strength (P) (ref. 131, concentration of organic modifier and denaturating agents (ref. 141, protein isolation source (ref. 151, stability of work and corrosive resistance of chromatographic system (ref. 16). The complicated choice of these and other parameters has been recently made with the help of computer expert system (ref. 17). According to the classical SEC, there should no interaction between the column support and substances, which are separated, however, in practice it is difficult to achieve a purely size-exclusion effect in the separation of components. Due to a great variety of chemical structures, usually found in protein specimens, and high heterogeneity of the compound, which is present in protein chains, several mechanism can be responsible for adsorption. There are several types of interaction, mainly of ion, electrostatic, hydrophobic and mixed mode, the prevention of which reduces non-exclusion influence (ref. 10). Note a: used abbreviations: HPLC high performance liquid chromatography, SEC size-exclusion chromatography.