[Recombinant human insulin. VII. Increased efficacy of chromatographic separation based on a principle of bifunctionality]. / Genno-inzhenernyi insulin cheloveka. VII. Povyshenie éffektivnosti khromatograficheskogo razdeleniia pri ispol'zovanii printsipa bifunktsional'nosti.

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. The dependences of the separation selectivity and resolution on the mobile phase composition and properties (a salt buffer type, pH, ionic strength) were found. It was demonstrated that the separation selectivity can be regulated by altering the contribution of each of the two separation mechanisms and the bifunctional sorbent used allows higher selectivity in the separation of close protein analogs than monofunctional sorbents.

[Genetic engineering of human insulin. IV. Development and optimization of an analysis system using reversed phase high pressure liquid chromatography]. / Genno-inzhenernyi insulin cheloveka. IV. Razrabotka i optimizatsiia sistemy analiza s pomoshch'iu obrashchenno-fazovoi vysokoéffektivnoi zhidkostnoi khromatografii.

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. A mechanism of the resolution and selectivity control is suggested.

[Separation of recombinant proteins by chromatography on vesicular sorbents]. / Razdelenie rekombinantnykh belkov s pomoshch'iu khromatografii na vezikuliarnykh sorbentov.

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. In the present study, separations of the recombinant proinsulin fusion protein oligomer and monomer, the recombinant human gamma-interferon monomer and dimer and recombinant tumour necrosis factor-alpha were investigated. For peak identification, the fractions and starting samples of the recombinant proteins were analysed by HPLC. The separations occurred without any sorption effects and with high efficiency and resolution of the protein peaks at a short column (10 cm). The VP is characterised by a high load ability, which favours the scale-up purification of the recombinant proteins. The combination of VP and HPLC is a considerable advance in biotechnology separation.

[Genetic engineering of human insulin. II. Exclusion HPLC of biotechnological precursors. Factors affecting resolution and selectivity]. / Genno-inzhenernyi insulin cheloveka. II. Ekskliuzionnaia VEZhKh biotekhnologicheskikh predshestvennikov. Faktory, vliiaiushchie na razreshenie i selektivnost'.

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]. / Genno-inzhenernyi insulin cheloveka. 1. VEZhKh v analize produktov osnovnykh stadii proizvodstva.

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. The authenticity of insulin obtained in the Shemyakin Institute of Bio-organic Chemistry by the scheme described in the present paper was confirmed by means of some physical and chemical methods and biological activity analysis.