[Recombinant human insulin. VII. Increased efficacy of chromatographic separation based on a principle of bifunctionality].

Retention mechanisms of insulin and deamido[AsnA21] insulin on the bifunctional sorbent Armsphere-C8(PR) in conditions of reversed-phase chromatography (HPLC and ion-pair HPLC) were studied. In accordance with the chemical differences of these proteins, molecular mechanisms of their interaction with silica gel modified with hydrophobic and ion-exchange groups were revealed. The possibility of simultaneous interaction of sorbed proteins with the stationary phase by both mechanisms under conditions of reversed-phase HPLC was demonstrated.

[Genetic engineering of human insulin. IV. Development and optimization of an analysis system using reversed phase high pressure liquid chromatography].

The effectiveness of the RP HPLC application for the step-by-step analysis of the recombinant insulin production was studied. Properties of a number of commercial and experimental columns in different chromatographic conditions were considered. A three-dimension optimization of selectivity and resolution versus pH and ion strength was carried out.

[Separation of recombinant proteins by chromatography on vesicular sorbents].

Vesicle chromatography, a recently developed method for separation of biomolecules, uses the vesicular packing (VP) material (clusters of microcapsules derived from plant cells), which was tested with respect to its application for the recombinant protein separation. Since VP has a well-defined separation limit, biomolecules are distributed in two separate peaks: large molecules are excluded and small molecules permeate through cell walls into the empty cell lumen. Recombinant proteins frequently form oligomers, which differ from monomers not only in size but also chemically and biologically.

[Genetic engineering of human insulin. II. Exclusion HPLC of biotechnological precursors. Factors affecting resolution and selectivity].

Mechanisms of the exclusion-sorption interaction of insulin-containing proteins with the column support in accordance with the chemical and three-dimensional structure were considered. The weak adsorption of the linear proinsulin and fusion protein in non-denaturing conditions, and dynamics of the SDS-protein complex's formation in denaturing conditions were studied. The obtained results are used for SE HPLC analysis of the main products and intermediates at essentially all steps of the recombinant human insulin production.

[Genetically engineered human insulin. 1. HPLC in analyzing products of basic production stages].

Application of some variants of HPLC for the step-by-step analysis of recombinant human insulin production was studied. Chromatographic columns with commercial and specially developed supports for size-exclusion, ion-exchange and reverse phase HPLC were used. Effective combinations of the chromatographic techniques for analysis of products and intermediates at every technological step were found and used for production of insulin.